Intranasal Administration

ABSTRACT

A delivery device for and method of modulating a condition relating to sodal cognition and/or behaviour in a human subject using oxytocin, non-peptide agonists thereof and/or antagonists thereof, comprising: providing a nosepiece to a first nasal cavity of the subject; and providing a supply unit for administering less than 24 IU of oxytocin, non-peptide agonists thereof and/or antagonists thereof through the nosepiece to an upper region posterior of the nasal valve which is innervated by the trigeminal nerve.

FIELD OF THE INVENTION

The present invention relates to the intranasal administration ofoxytocin (OT), especially for the modulation of social cognition and/orbehavior, being mental and/or behavioral operations underlying socialinteractions, and also to the intranasal administration of otherpeptides, including Orexin-A, especially for the treatment ofnarcolepsy, and insulin, especially for the treatment of diabetes.

BACKGROUND OF THE INVENTION

A growing body of evidence demonstrates a role of OT in social cognitionand behavior¹⁻³. For instance, a single administration of OT hasincreased empathy⁴⁻⁵, trust⁶, group-serving behaviours⁷⁻⁸, sensitivityof eye gaze⁹, and theory-of-mind performance in healthy individuals¹⁰and in patients with psychiatric disorders¹¹. OT has also been proposedas a novel therapy for disorders characterized by social dysfunction,such as autism and schizophrenia spectrum disorders¹²⁻¹³,

Despite initial promise, however, recent work has either failed toidentify changes in social behavior after OT administration¹⁴ or hasprovided results that are only significant for specific subgroups orcontexts¹⁵. These mixed results have been largely attributed to suchcontextual and individual differences¹⁶, and factors that may influencebiological activity of exogenous OT have yet to be thoroughlyinvestigated¹⁵⁻¹⁸.

The present inventors postulate that other factors to dose and deliverymethod may influence biological activity of exogenous OT, and similarlyto other peptides, including Orexin-A and insulin.

Olfactory nerve fibres innervate a limited segment of the deep uppernarrow nasal passage, while the trigeminal nerve provides sensory andparasympathetic innervation to the deep upper and posterior segments ofthe nose. Drug transport along these cranial nerve fibres may offer apotential direct route to the central nervous system (CN)^(15,23)circumventing the blood-brain barrier (BBB), and this segment is notadequately targeted by conventional nasal spray devices^(15,26).

The present inventors postulate that, by virtue of nose-to-brainactivity, the targeted intranasal administration of OT to thisinnervated segment of the nasal passage could enable pharmacodynamiceffects in the brain disproportionate to what would be achieved byabsorption into the blood, and that this method of targeted delivery mayimprove the reliability, therapeutic index, and effect magnitude of OTtreatment effects due to improved drug deposition^(15,31-32).

An unchallenged assumption in the literature that would benefit fromcloser experimental scrutiny in humans is that intranasal administrationis the best means of delivering OT to modulate social cognition andbehaviour¹⁵.

Despite early work demonstrating that intravenous (IV) administrationcan influence social behavior and cognition³³⁻³⁴—presumably via bloodabsorption and subsequent action across the BBB—subsequent human studiesassessing the effect of OT on cognitive functions have used methods thatdeliver OT via the nasal cavity. Although there is a strong theoreticalbasis that intranasal delivery is a more appropriate means ofadministering OT, a controlled comparison of pharmacodynamics (PD)effects after intranasal (i.e., nose-to-brain) and intravenoustransportation across the BBB) administration has not been done.

Furthermore, in relation to the dosing regimen, the majority ofintranasal OT studies have evaluated between 20 and 40 internationalunits (IU)³⁶, There is no comprehensive empirical evidencesubstantiating this dosage³⁷⁻³⁸, though successful in other disciplines(e.g. obstetrics)³⁹, This is despite the negative long-term effects ofOT treatment observed in non-human adolescent mammals⁴⁰, and thepresence of OT and cross-reactive vasopressin (AVP) receptors throughoutthe body⁴¹ that are involved in a variety of homeostatic functionsrelated to observed side effects⁴².

It is an aim of the present invention to provide for improved efficacyin the intranasal administration of oxytocin (OT), especially for themodulation of social cognition and/or behavior, and other peptides,including Orexin-A, especially for the treatment of narcolepsy, andinsulin, especially for the treatment of diabetes.

SUMMARY OF THE INVENTION

In one aspect the present invention provides a method of modulatingconditions relating to social cognition and/or behaviour in a humansubject using oxytocin, non-peptide agonists thereof and/or antagoniststhereof, comprising: providing a nosepiece to a first nasal cavity ofthe subject; and administering less than 24 IU of oxytocin, non-peptideagonists thereof and/or antagonists thereof to an upper region posteriorof the nasal valve which is innervated by the trigeminal nerve.

In another aspect the present invention provides a method of modulatinga condition in a human subject using a peptide, non-peptide agoniststhereof and/or antagonists thereof, comprising: providing a nosepiece toa first nasal cavity of the subject; and administering less than 24 IUof a peptide, non-peptide agonists thereof and/or antagonists thereofthrough the nosepiece to an upper region posterior of the nasal valvewhich is innervated by the trigeminal nerve.

In a further aspect the present invention provides a nosepiece fordelivering substance to a nasal cavity of a subject, the nosepiececomprising: a first, inner body part; and a second, outer body partwhich is disposed about at least a distal portion of the inner body partand defines a tip; wherein the inner body part comprises a base portionwhich defines a flow passage therethrough, and a projection at thedistal end thereof which supports the tip and confers a rigidity in thesagittal direction, which enables the tip to open fleshy tissue at anupper region of the nasal valve and thereby expand an open area of thenasal valve, and a flexibility in a lateral direction, orthogonal to thesagittal plane, which facilitates insertion of the tip into the nasalvalve.

In a yet further aspect the present invention provides a nosepiece fordelivering substance to a nasal cavity of a subject, the nosepiececomprising a body part which comprises a base portion which defines aflow passage therethrough, and a projection at a distal end of the baseportion which at least in part provides a tip of the nosepiece andconfers a rigidity in the sagittal direction, which enables the tip toopen fleshy tissue at an upper region of the nasal valve and therebyexpand an open area of the nasal valve, and a flexibility in a lateraldirection, orthogonal to the sagittal plane, which facilitates insertionof the tip into the nasal valve.

DESCRIPTION OF THE FIGURES

Preferred embodiments of the present invention will now be describedhereinbelow by way of example only with reference to the accompanyingdrawings, in which:

FIGS. 1(a) to (c) illustrate a delivery device in accordance with oneembodiment of the present invention;

FIGS. 2(a) to (e) illustrate perspective, lateral, front, underneath andlongitudinal sectional views (along section A-A) of the nosepiece of thedevice of FIGS. 1(a) to (c);

FIGS. 3(a) to (e) illustrate perspective, lateral, front, underneath andlongitudinal sectional views (along section B-B) of the inner body partof the nosepiece of the device of FIGS. 1(a) to (c);

FIG. 4 illustrates the social-cognitive task design of the study;

FIGS. 5(a) to (f) represent mean emotional ratings by stimulus, beingangry ratings of ambiguous faces (5(a)), happy ratings of ambiguousfaces (5(b)), happy ratings of happy faces (5(c)), and ratings of happyfaces (5(d)), angry ratings of angry faces (5(e)) and happy ratings ofangry faces (5(f)), and treatment, being the intranasal administrationof 8 IU of OT (81U-OT), the intranasal administration of 24 IU of OT (24IU-OT), the intravenous delivery of lIU of OT (IV-OT), and theintranasal administration of a placebo formulation (Placebo);

FIG. 6(a) represents the percentage reduction of anger ratings after the8 IU-OT administration as compared to Placebo by stimuli categories;

FIG. 6(b) represents the percentage reduction of anger ratings after the8 IU-OT administration as compared to the 24 IU-OT administration bystimuli categories;

FIG. 7(a) represents the mean OT plasma concentration over time afterthe administrations of 8 IU-OT, 24 IU-OT, IV-OT and Placebo, with errorbars representing standard error of the mean;

FIG. 7(b) represents the mean vasopressin (AVP) plasma concentrationover time after the administrations of 8 IU-OT, 24 IU-OT, IV-OT andPlacebo, with error bars representing standard error of the mean;

FIG. 7(c) represents the mean cortisol plasma concentration over timeafter the administrations of 8 IU-OT, 24 IU-OT, IV-OT and Placebo, witherror bars representing standard error of the mean;

FIG. 8 illustrates the relationship between the mean nasal valvecross-sectional area and angry ratings of neutral faces by subjectsafter the administrations of 8 IU-OT, 24 IU-OT, IV-OT and Placebo;

FIG. 9(a) illustrates time-course spatial maps determined from fMRIanalysis for Independent Component #37 showing strong amygdala, medialtemporal lobe (MTL) and brain stem weighting;

FIG. 9(b) illustrates time-course spatial maps of the two largestclusters (voxel-wise p<0.01, uncorrected) in Independent Component #37,which are localized within the left and right amygdala, respectively;

FIG. 9(c) illustrates time-course spatial maps of the two largestclusters showing significantly (p<0.05, cluster size corrected usingpermutation testing) increased connectivity in the 8 IU-OT treatment ascompared to Placebo in the left and right amygdala, respectively;

FIGS. 10(a) and (b) illustrate boxplots of the mean connectivity withinthe two clusters from fMRI analysis showing significant (p<0.01,uncorrected) main effects of the OT condition;

FIGS. 11(a) and (b) illustrate boxplots of the mean connectivity withinthe two clusters from fMRI analysis showing significantly (p<0.05,cluster size corrected) increased connectivity after the 8 IU-OT andPlacebo treatments;

FIGS. 12(a) and (b) represent, by way of spaghetti plots, theconnectivity values in all conditions in each of the significantamygdala clusters obtained from the pairwise comparison of the 8 IU-OTand Placebo treatments for each individual;

FIG. 13(a) illustrates violin plots which represent right amygdalaactivation and box and whisker plots which represent the median and 50%interquartile ranges after the administrations of 8 IU-OT, 24 IU-OT,IV-OT and Placebo;

FIG. 13(b) illustrates the main effect of the presentation of facesacross emotions and 8 IU-OT, 24 IU-OT, IV-OT and Placebo treatments; and

FIGS. 14(a) to (c) represent the relationship between mean pupildiameter and right amygdala activity after the 8 IU-OT treatment whileprocessing angry, ambiguous and happy facial stimuli.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Device

FIGS. 1(a) to (c) illustrate a manually-actuated nasal delivery devicein accordance with one embodiment of the present invention,

The delivery device comprises a housing 115, a nosepiece 117 for fittingin a nasal cavity of a subject, a mouthpiece 119 into which the subjectin use exhales, such as to enable delivery of an air flow into andthrough the nasal airway of the subject on exhalation by the subjectthrough the mouthpiece 119, and a delivery unit 120, which is manuallyactuatabie to deliver substance to the nasal cavity of the subject.

The housing 115 comprises a body member 121, in this embodiment ofsubstantially elongate, tubular section which includes an aperture 123at one end thereof, through which projects an actuating part of thedelivery unit 120, in this embodiment as defined by the base of asubstance-containing chamber 173 of a substance-supply unit 169.

The housing 115 further comprises a valve assembly 127 which is fluidlyconnected to the nosepiece 117 and the mouthpiece 119, and operablebetween closed and open configurations, as illustrated in FIGS. 3 and 4,such as to provide for an air flow, in this embodiment in the form of aburst of air, through the nosepiece 117 simultaneously with actuation ofthe delivery unit 120, as wili be described in more detail hereinbelow.

The valve assembly 127 comprises a main, body element 128 which includesa valve seat 129 defining a valve opening 130, and a valve element 131which is movably disposed to the body element 128 between closed andopen positions, as illustrated in FIGS. 1(b) and (c).

As particularly illustrated in FIG. 1(c), the body element 128 comprisesa pivot 135, in this embodiment to one, lower side of the valve seat129, to which one end 145 of the valve element 131 is pivoted, and asliding surface 137, in this embodiment to the other, upper side of thevalve seat 129, against which the other end 147 of the valve element 131is slideabie.

The valve element 131 comprises an elongate arm 141, in this embodimenta flexible arm, one end 145, in this embodiment the lower end, of whichis pivoted to the pivot 135 of the body element 128, and the other,upper end 147 of which slideably engages the sliding surface 137 of thebody element 128, and a valve member 149 which is supported by the arm141.

In this embodiment the arm 141 comprises a first, here lower, armsection 151, which is biased, here inwardly, such that, when the valveelement 131 is in the closed, rest position, the lower arm section 151is inclined inwardly relative to the longitudinal axis of the housing115 and engageable by the substance-supply unit 169 when manuallyactuated to move the valve element 131 to the open position, as will bedescribed in more detail hereinbelow.

In this embodiment the arm 141 further comprises a second, here upper,arm section 153, which engages the sliding surface 137 of the bodyelement 128 and acts to bias the valve element 131 to the closedposition.

In this embodiment the valve member 149 comprises a seal 161 in thisembodiment a flexible or resilient element, which acts to close thevalve opening 130 as defined by the valve seat 129 when the valveelement 131 is in the closed position, and a support 163 which supportsa central region of the seal 161.

With this configuration, where the seal 161 is centrally supported, whenthe valve element 131 is moved to the open position, the support 163biases the central region of the seal 161, causing the seal 161 to bulgeoutwardly in this central region and thus provide that the seal 161engages the valve seat 129 only at the peripheral edge of the seal 161,until the point is reached when the seal 161 is suddenly and explosivelyreleased from the valve seat 129.

This mode of release is believed to be particularly effective in thepresent application where it is desired to achieve a sudden, initialburst of air flow, in that substantially the entire sealing surface ofthe seal 161 is released in one instant, which compares to analternative mode of a peeling-type release, where a smaller section of asealing surface is released, followed by the remainder of the sealingsurface, which tends to provide a smaller initial burst pressure.

In this embodiment the delivery unit 120 comprises an outlet unit 167for delivering substance into the nasal airway of the subject, and asubstance-supply unit 169 for delivering substance to the outlet unit167.

In this embodiment the outlet unit 167 comprises a nozzle 171 fordelivering substance to the nasal airway of the subject. In thisembodiment the nozzle 171 is configured to provide an aerosol spray. Inan alternative embodiment, for the delivery of a liquid, the nozzle 171could be configured to deliver a liquid jet as a column of liquid.

In a preferred embodiment the distal end of the outlet unit 167 isconfigured to extend at least about 2 cm, preferably at least about 3cm, and more preferably from about 2 cm to about 3 cm, into the nasalcavity of the subject.

In this embodiment the substance supply unit 169 is a pump unit, whichcomprises a substance-containing chamber 173 which contains substanceand extends from the aperture 123 in the housing 115 as the actuatingpart of the substance-supply unit 169, and a mechanical delivery pump175 which is actuatable, here by depression of the substance-containingchamber 173, typically by a finger or thumb of the subject, to deliver ametered dose of substance from the substance-containing chamber 173 tothe outlet unit 167 and from the nozzle 171 thereof, here as an aerosolspray.

In this embodiment the substance-containing chamber 173, when depressedto actuate the substance supply unit 159, engages the lower arm section151 of the arm 141 of the valve element 131, such as simultaneously toprovide for actuation of the substance-supply unit 169 and opening ofthe seal 161 of the valve element 131, whereby substance, here in theform of a spray, and an air flow, here as a burst of air, aresimultaneously delivered to the nasal cavity of the subject.

In this embodiment the mechanical delivery pump 175 is a liquid deliverypump for delivering a metered dose of substance.

In this embodiment the substance-supply unit 169 is a multi-dose unitfor delivering a plurality of metered doses of substance in successivedelivery operations.

In this embodiment the housing 115 further comprises a sealing member181, here an annular seal, in the form of an O-ring, which slideablyreceives the substance-containing chamber 173 of the substance-supplyunit 169, such as to prevent the escape of the delivered air flow fromthe aperture 123 in the housing 115,

FIGS. 2(a) to (e) and 3(a) to (e) illustrate the nosepiece 117 of thedescribed embodiment.

As particularly illustrated in FIG. 2(e), the nosepiece 117 is formed oftwo body parts 202, 204, a first, inner body part 202, here formed of aplastics material, and a second, outer body part 204, here formed of asofter, resilient material, such as a rubber or elastomeric material,which is disposed about the distal end of the inner body part 202 anddefines a tip element 206,

In this embodiment the inner body part 202 is formed of an acrylonitrilebutadiene styrene (ABS) plastic, here Guardian/Lustran® ABS 308 (assupplied by Ineos ABS (USA) Corporation),

In this embodiment the outer body part 204 is formed of a thermoplasticelastomer (TPE), here Versaflex® OM 1040X1 (as supplied by GLS/PolyOneCorporation), having a Shore A hardness of 42.

As particularly illustrated in FIGS. 3(a) to (e), in this embodiment theinner body part 202 comprises a base portion 208 which defines a flowpassage 209 therethrough, and a projection 112 at the distal,forwardmost end thereof which supports the tip 106 of the nosepiece 117.

In this embodiment the distal, forwardrnost end of the base portion 208defines a surface 210 which tapers or is inclined in relation to thelongitudinal axis of the nosepiece 117, such that the surface 210 of thebase portion 208 is inclined in an direction away from the distal end ofthe projection 212, and the base portion 208 is shorter at that sidewhich is opposite to the projection 212.

The projection 112 is configured to confer a rigidity in the sagittaldirection, which enables the tip 206 of the nosepiece 117 to open thefleshy tissue at upper region of the nasal valve and thereby expand theopen area of the nasal valve, and a flexibility in the lateraldirection, which facilitates insertion of the tip 206 of the nosepiece117 into the nasal valve, In this embodiment, from measurement byacoustic rhinometry (AR), the nosepiece 117 provides for expansion ofthe area of the nasal valve to an area which is at least twice the areaof the nasal valve when unexpanded and in a rest state.

In this embodiment the projection 212 extends axially in substantiallyparallel relation to the longitudinal axis of the nosepiece 117.

In this embodiment the projection 212 has the form of a blade, with alength dl in the sagittal direction being greater than a length d2 inthe lateral direction,

In this embodiment the length d1 in the sagittal direction is 1.5 timesgreater than the mean length d2 in the lateral direction.

In one embodiment the length d1 in the sagittal direction is 1.7 timesgreater than the mean length d2 in the lateral direction.

In this embodiment the length d1 in the sagittal direction is 1.9 timesgreater than the mean length d2 in the lateral direction.

In this embodiment the length d1 in the sagittal direction is 2 timesgreater than the mean length d2 in the lateral direction,

In this embodiment the projection 212 has a length d1 in the sagittaldirection of about 2 mm.

In this embodiment the projection 212 has a length d2 in the lateraldirection of about 1 mm.

In this embodiment the projection 212 has a main body section 214 and atip section 216 which has a shorter length d3 in the sagittal directionthan the length d1 of the main body section 214, here defining a step atan inner edge thereof.

In this embodiment the projection 212 has a tapering lateralcross-section along its length, with the length d2 in the lateraldirection reducing in cross-section along its length towards the distalend.

In this embodiment the length d2 in the lateral direction reduces fromabout 1.1 mm to about 0.8 mm from the proximal to the distal end of theprojection 212.

Study

A randomized, double-blind, double-dummy, crossover study was performed,in which 18 healthy male adults were randomly assigned, and 16 completedfour single-dose treatments; these being (1) the intranasaladministration of a liquid spray of 8 IU of OT delivered using thedevice of FIGS. 1(a) to (c) (hereinafter 8 IU-OT), (2) the intranasaladministration of a liquid spray of 24 IU of OT delivered using thedevice of FIGS. 1(a) to (c) (hereinafter 24 IU-OT), (3) the intravenousdelivery of HU of OT (hereinafter IV), and (4) the intranasaladministration of a liquid spray of a placebo using the device of FIGS.1(a) to (c) (hereinafter Placebo).

This study compared pharmacodynamic (PD) effect of OT on socialcognition and behavior, as indexed by the presentation of emotionalstimuli and in particular amygdala activity.

In order to examine the neural correlates of OT's behavioral andcognitive effects, researchers have adopted brain-imaging tools such asfunctional magnetic resonance imaging (fMRI). Converging evidence fromthis field suggests the amygdala, a key brain region for emotionregulation⁸⁶, processing⁸⁷ and detection¹¹³, is an important target ofOT administration, The modulation of amygdala activity in response toemotional stimuli is arguably the most replicated and well-characterizedresult within brain imaging and intranasal OT studies^(88,89,114-117).Irrespective of this prior work, however, it is not dear how OT travelsto the brain or which OT dose is more likely to modulate the recruitmentof amygdala during the presentation of emotional stimuli. By, comparingamygdala activity after both intranasal and intravenous OTadministration, when comparable blood levels are achieved, research candetermine if neural modulation occurs via direct nose-to-brain transport(as currently assumed) or through systemically circulating OT crossingthe BBB. There is both animal⁷⁰ and human.³³⁻³⁴ research to suggestsystemic OT can influence social behavior and cognition—however,research has not yet evaluated amygdala activity after intravenousdelivery with an intranasal OT comparator.

Recent theories also underscore OT's role in the facilitation ofapproach-related behaviours¹¹⁸ and the modulation of social stimulisalience¹⁶. Given the established relationship between cognitiveresource allocation and pupil dilation¹¹⁹⁻¹²⁰, pupilometry offers anon-invasive neurobiological measure of engagement towards emotionalstimuli. Research indicates that intranasal OT enhances pupil dilation⁵⁵and the salience of social cues¹²¹. However, the relationship betweenamygdala activity and pupil-indexed cognitive engagement has yet to beexplored and may contribute to a better understanding of the effects ofOT.

Primary outcomes were the evaluation of facial emotional expression, inparticular in relation to amygdala activity, and secondary outcomesincluded pharmacokinetic (PK) profiles and ratings of trustworthiness,

This study hypothesized a main effect of the administration of 8 IU-OTand 24 IU-OT on the perceived intensity of anger, and that this effectwould be more pronounced with ambiguous emotional stirnuli compared tostimuli with less ambiguous emotional expressions.

This study examined dose-dependent effects of 8 IU-OT and 24 IU-OT.

This study also investigated the impact of OT on trust ratings of thesame facial stimuli.

In order to characterize PK and evaluate potentially differentrelationships between PK and PD by method of drug delivery, the timecourse of blood plasma concentrations of OT and physiologicallyinteracting substances vasopressin (AVP) and cortisol were measuredfollowing treatment. Modulation of social cognition after 8 IU-OT and 24IU-OT administration, but not after IV-OT producing comparable bloodexposure, would provide evidence that 8 IU-OT and 24 IU-OTadministration is, at least in part, directly acting on the brain ratherthan across the BBB.

Eligible participants were males between the ages of 18 to 35, in goodphysical and mental health, Exclusion criteria included use of anymedications within the last 14 days, history of alcohol or drug abuse,clinically relevant history of physical (including renal, cardiac,endocrine, pulmonary, hepatic, nervous, gastrointestinal, hematologicaland metabolic disorders), or psychiatric illness, and IQ<75. Fifty-sevenmale volunteers were assessed for eligibility, and 18 participants wereselected aged 20-30 years (M=23.81, SD=3.33). Two participants withdrewafter enrollment [1 withdrew after the first session, and the otherwithdrew after completing three sessions], and data from theseparticipants is not included in the analyses.

A screening visit occurred between 3-21 days prior to randomization. TheWechsler Abbreviated Scale of Intelligence⁵² and the Mini-InternationalNeuropsychiatric Interview⁵³ were used to index IQ and confirm theabsence of psychiatric illness, respectively. A physical examination wasperformed, including ECG and the collection of routine blood samples. Inaddition, an otolaryngologist confirmed normal nasal anatomy and patencyin participants (via physical examination) and acoustic rhinometry (AR)data were collected (SRE 2000; Rhinometrics, Lynge, Denmark). Threemeasures were calculated from the AR data: Minimum cross-sectional area(MCA; i.e., the narrowest section of the nasal cavity), total volumefrom nostril to 5 cm deep (TV0-5), and total volume from 2-5 cm deep(TV2-5).

A randomized, placebo-controlled, double-blind, double-dummy,four-period crossover design was used for this study. Participants wererandomized to one of four treatment sequences, using a four-periodfour-treatment Latin square method (ACDB—BDCA—CBAD—DA BC in a 4:4:4:4ratio), with a period of at least six days between treatments to preventpotential carry-over effects. Both the participants and research teamwere blinded to treatment using visually matching devices and IVapparatus during data collection.

In this study, the delivery device capitalizes on two aspects of nasalanatomy to facilitate delivery to the respiratory and nasal epithelia³².Firstly, as the user is blowing through the mouth against a resistance,the soft palate automatically closes, isolating the nasal cavity fromthe oral cavity, preventing lung deposition and limitinggastrointestinal deposition²³. Secondly, in conjunction with closure ofthe soft palate, an optimized nosepiece is employed that allows deeperinsertion to directs the exhaled breath and OT into the upper-posteriornasal cavity segments²³.

The 8 IU-OT, 24 IU-OT and Placebo formulations were supplied bySigma-Tau Industrie Farmaceutiche Riunite S.p.A.. The Placeboformulation was 0.9% sodium chloride.

The IV-OT formulation was supplied by AS Grindeks, Riga, Latvia wassupplied as a 10 IU/m1 formulation and added to a 0.9% sodium chloridesolution for infusion shortly before administration (600 ml/hour over 20minutes). The intravenous dosage and infusion rate was chosen so as togenerate peripheral OT concentrations that are equivalent to intranasaldelivery, as confirmed by experiment.

In order to ensure appropriate use and standardization, participantswere trained on the use of the intranasal delivery device by watching ademonstration video, following written instructions, and administeringpractice saline sprays under the supervision of trained research staffduring the screening session.

At the beginning of each experimental session, exclusion and inclusioncriteria were confirmed and the State-Trait Anxiety Inventory 54 wasadministered. Blood samples were taken to assess routine measures andacoustic rhinornetry (AR) was performed (per procedures duringscreening) to confirm that the nasal cavity environment did notsignificantly differ between sessions due to nasal cycles²⁴.

Participants completed the social-cognitive task 40 minutes aftertreatment in a magnetic resonance imaging (MRI) scanner while functionalMRI and physiology data was recorded.

Participants were presented with visual stimuli through MRI-compatiblegoggles (VisualSystem; NordicNieuroLab, Bergen, Norway) using E-Prime2.0 (Psychology Software Tools, PA, USA), and responded using a gripresponse collection system (ResponseGrip, NordicNeuroLab, Bergen,Norway).

Participants were presented with 20 male and 20 female faces⁵⁵displaying angry, happy, and emotionally ambiguous facial expressions[derived from the Karolinska Directed Emotional Faces database⁵⁶] and 20images of geometrical shapes. The social-cognitive task consisted offive blocks of 20 trials, as illustrated in FIG. 4. Each trial ofapproximately 140 s duration comprised the following sequence: Fixationcross of 3 s duration→Stimulus (face/shapes) presentation of 1 sduration→Q1 of 3.25 s duration (maximum response window)→Q2 of 3.25 sduration (maximum response window).

For the evaluation of the faces, participants were asked a firstquestion (Q1) which was either: How angry is this person? (anchors: notangry—very angry) or, How happy is this person? (anchors: not happy—veryhappy), and a second question (Q2), which was always the same: How muchwould you trust this person? (anchors: not at all—very much). For bothquestions, participants were asked to rank their answer on a visualanalogue scale (VAS) from 1 to 5, with location of the cursor on the VASrandomized on the presentation of each question, Mean ratings for eachof the questions were averaged per session within each of the emotionalcategories, yielding seven behavioral variables (Q1: Happy face—happy,Happy face—angry, ambiguous face—happy, ambiguous face—angry, angryface—happy, angry face—angry; Q2; Trust). These stimuli and questionswere chosen to assess three levels of emotion perception; ambiguous,non-ambiguous with corresponding cues and ratings (e.g,, angry ratingson angry ratings), and non-ambiguous with conflicting cues and ratings(e.g., angry ratings of happy faces).

For the evaluation of the shapes, participants were asked either: (Q1)How yellow is this shape? (anchors: not yellowvery yellow) or How blueis this shape? (anchors: not yellow—very yellow), Q2 was always: Howmuch do you like this color? (anchors: not at all—very much). In thesame manner as for ranking the faces, participants were asked to ranktheir answer on a visual analogue scale (VAS) from 1 to 5, with locationof the cursor on the VAS randomized on the presentation of eachquestion.

Brain imaging data was collected on a 3T General Electric Signa HDxtscanner with an 8-channel head coil (GE Healthcare, Milwaukee, Wis.,USA).

In the acquisition of MRI data, the protocol included a T2*-weightedgradient echo-planar imaging (EPI) sequence acquired in the transverseplane with the following parameters: Repetition time (TR)=2400ms, echotime (TE)=30 ms, flip angle (FA)=90°, 64×64 matrix, One run of 528volumes was collected for each individual in each OT condition (48slices; in-plane resolution 3.75×3.75 mm; slice thickness 3.2 mm, nogap). A T1-weighted volume, used for co-registration purposes, wasacquired using a sagittal fast spoiled gradient echo (FSPGR) sequencewith the following parameters: TR=7.8 ms, TE=2.9 ms, FA=12°, 166 slices;in-plane resolution: 1×1, slice thickness: 1.2 mm, 255×256 matrix.

Pupilometry data was collected using an MR-compatibie coil-mountedinfrared EyeTracking system (NNL EyeTracking camera®, NordicNeuroLab,Bergen, Norway) at a sampling rate of 60 Hz. Data was recorded using theiView X Software (SensoMotoric Instruments, Teltow, Germany), with atrigger from the stimulus computer syncing the onset of the pupilometryrecording to stimulus presentations,

During the experimental sessions, blood samples were collected via IVcatheter to assess peripheral levels of OT, AVP, and cortisol atbaseline and five time points after the completion of the 20-minute IVadministration (0 mins, 10 mins, 30 mins, 60 mins, and 120 mins)throughout the session. Blood samples were centrifuged at 4° C. within20 minutes of blood draw, after which plasma was frozen at −80° C. untilenzyme-linked immunosorbent assay (ELISA) using commercially availablekits (Enzo Life Sciences, Farmingdale, N.Y.) was performed usingstandard techniques (including sample extraction).

Pharmacodynamic Aralysis

Analysis was conducted using IBM SPSS Statistics version 22 (IBM Inc.)to determine pharmacokinetics and examine the impact of treatment onoutcome measures. A linear mixed-model (LMM) approach was adopted⁵⁸,congruent with a recent intranasal crossover psychotropic drug trial⁹⁵,for the analysis of emotional expression evaluation, pharmacokinetics,state anxiety, and trustworthiness. All models were fitted using anunstructured matrix. For any significant main effects (i.e., p<0.05),post-hoc tests were performed with the adjustment of critical p valuesto correct for multiple comparisons using a 5% false discovery rate(FDR)⁵⁹.

Experimental treatment was both a fixed and repeated effect in a LMM toassess the impact of treatment on emotion and trustworthiness ratings.

Additionally, in order to investigate the impact of treatment on bloodplasma OT, AVP, cortisol concentration and state anxiety a LMM wasfitted with 3 fixed factors (treatment, time, treatment×time), 1repeated factor (treatment). In order to investigate if nasalenvironments changed between treatment conditions, a repeated measuresMANOVA was performed with three dependent variables; MCA, TV0-5, andTV2-5.

Participant responses to the task are presented in Table 1. Due toequipment difficulties, data was not collected during two (out ofsixty-four) testing sessions. A LMM revealed a significant main effectof treatment in the ratings of anger when presented ambiguous faces[F(3,14.72)=7.62, p=0.003; FIG. 5(a)]. Follow-up pairwise comparisons(q=0.05, revised critical value of p<0.017) indicated that angry ratingsfor ambiguous faces were significantly reduced in the 8 IU-OT treatmentcondition in comparison to both Placebo (p=0.011; mean decrease=17%, SEdecrease 6%) and 24 IU-OT (p=0.003; mean decrease=17%, SE decrease 5%)treatments, There were no main effects of treatment observed for otheremotional categories or trustworthiness ratings (FIGS. 5(b) to (f)).

TABLE 1 Participant ratings in the social cognition task Outcomes Linearmixed-model main effect Emotional expression evaluation 8 IU-OT 24 IU-OTIV-OT Placebo df F p Angry ratings of ambiguous faces 2.11 (0.15) 2.46(0.17) 2.32 (0.18) 2.41 (0.15) 3, 14.72 7.62 0.003 Happy ratings ofambiguous faces 2.61 (0.14) 2.67 (0.12) 2.38 (0.14) 2.51 (0.13) 3, 15.171.78 0.193 Angry ratings of angry faces 3.51 (0.2)  3.54 (0.16) 3.68(0.2)  3.57 (0.16) 3, 14.76 0.82 0.505 Happy ratings of angry faces 4.15(0.62) 4.26 (0.57) 4.29 (0.54)  4.3 (0.36) 3, 15   0.32 0.314 Angryratings of happy faces 1.23 (0.02) 1.25 (0.02) 1.24 (0.02) 1.24 (0.02)3, 15   0.97 0.433 Happy ratings of happy faces 4.11 (0.16) 4.26 (0.14)4.31 (0.13)  4.3 (0.09) 3, 13.84 1.32 0.309 Trustworthiness 3.13 (0.04)3.15 (0.05) 3.16 (0.05) 3.11 (0.03) 3, 14.27 2.57 0.095 Note. Unlessspecified otherwise, values are estimated means based on linear mixedmodels with standard error in parenthesis.

In order to evaluate the specificity of the effect for ambiguous faces(vs. non-ambiguous faces with corresponding cues and non-ambiguous withconflicting cues), a percentage change score was calculated comparingratings after 8 IU-OT and Placebo treatments, and comparing 8 IU-OT with24 IU-OT treatments (i.e., the treatment comparisons that demonstratedsignificant differences in emotional ratings). Ambiguous=anger ratingsof ambiguous faces; NA−corresponding=Anger ratings of non-ambiguousfaces with corresponding cues; NA−conflicting=Anger ratings ofnon-ambiguous faces with conflicting cues. Stimuli category was both afixed and repeated effect in a LMM to assess the impact of stimulicategory on the reduction of anger ratings. For the LMM comparingpercentage change between the 8 IU-OT and Placebo treatment, there was amain effect for stimuli type [F(2,14.42)=4,79, p=0.025; FIG. 6(a)].Follow-up pairwise comparisons to the ambiguous stimuli category(q=0.05, revised critical value of p<0.025) indicated that thepercentage reduction of anger ratings of ambiguous stimuli wassignificantly reduced in comparison to the non-ambiguous(NA)/conflicting stimuli (p=0.012). For the LMM comparing percentagechange between the 8 IU-OT and 24 IU-OT treatment, there was a maineffect for stimuli type [F(2,14.05)=7.01p=0.007; FIG. 6(b)]. Follow-uppairwise comparisons to the ambiguous stimuli category (q=0.05, revisedcritical value of p<0.025) indicated that the percentage reduction ofanger ratings of ambiguous stimuli was significantly reduced incomparison to the non-ambiguous/conflicting stimuli (p=0.008).

Out of 384 possible data points, 19 OT, 26 AVP and 18 cortisol plasmaconcentration assessments were excluded due to technical issues relatingto blood sample collection or analysis.

Oxytocin blood plasma concentration: The mean OT plasma concentrationsover time after the administration of 8 IU-OT, 24 IU-OT, IV-OT andPlacebo (with error bars representing standard error of the mean) arerepresented in Table 2 and FIG. 7(a). For the 4 (treatment)×6 (time)LMM, there was a significant main effect of treatment on OT blood plasmaconcentration [F(3,88.71)=4.25, p=0.007]. Follow-op pairwise comparisons(q=0.05, revised critical value of p<0.025) revealed that plasma OTconcentration was significantly increased in the IV-OT (p=0.009), 8IU-OT (p=0.001), and 24 IU-OT (p=0.002) treatments compared to thePlacebo treatment. None of the other pairwise comparisons reachedsignificance, There was also a significant main effect for time[F(5,90.29)=5.93, p<0.001], with follow-up pairwise analyses (q=0.05,revised critical value of p<0.017) indicating significantly increasedplasma OT immediately after IV administration in comparison to baseline(p<0.001), 10 minutes (p=0.01), 30 minutes (p=0.001), 60 minutes(p=0.001), and 120 minutes after the completion of IV administration(p<0.001). There was no significant condition x time interaction,F(15,88.69)=1, p=0.461.

TABLE 2 8 IU-OT 24 IU-OT IV-OT Placebo Time Mean SEM N Mean SEM N MeanSEM N Mean SEM N −20 4.59 1.98 16.00 7.72 2.40 15.00 6.02 1.55 16.003.95 0.45 15.00 0 6.88 1.43 15.00 14.20 3.64 14.00 25.64 3.98 16.00 5.141.18 14.00 10 8.29 2.90 14.00 11.98 2.81 15.00 10.79 2.75 15.00 4.250.66 15.00 30 9.88 3.63 16.00 8.47 1.96 15.00 6.99 1.77 16.00 5.26 1.3914.00 60 9.76 2.63 16.00 7.70 1.98 16.00 9.50 2.44 14.00 5.02 1.06 15.00120 8.84 2.22 16.00 6.31 1.19 16.00 6.13 1.09 16.00 5.39 1.63 15.00

Vasopressin blood plasma concentration: The mean AVP plasmaconcentrations over time after the administration of 8 IU-OT, 24 IU-OT,IV-OT and Placebo (with error bars representing standard error of themean) are represented in Table 3 and FIG. 7(b). For the 4 (treatment)×6(time) LMM, there was a significant main effect of treatment on AVPblood plasma concentration [F(3,82,42)=4.55, p=0.005], Follow-uppairwise comparisons (q=0.05, revised critical value of p<0.0083)revealed plasma AVP concentration was significantly decreased after 24IU-OT treatment in comparison to Placebo treatment (p=0.008) and IV-OT(p−0.013), and significantly decreased after 8 IU-OT treatment incomparison to IV-OT (p=0.023), There was no significant main effect oftime [F(5,90.63)=1.81 p=0.12] or treatment×time interaction,F(15,82,46)=1.03, p=0.434.

TABLE 3 8 IU-OT 24 IU-OT IV-OT Placebo Time Mean SEM N Mean SEM N MeanSEM N Mean SEM N −20 4.76875 0.9233417 16 3.578571 0.5601391 14 11.725.314878 15 4.366667 0.7328786 15 0 3.185715 0.4818528 14 3.1285710.46639 14 4.906667 1.317554 15 3.86 0.707228 15 10 2.876923 0.464392913 3.107143 0.4459173 14 4.471428 1.028999 14 3.49375 0.5508682 16 303.0875 0.4738033 16 2.471428 0.3692029 14 4.085714 0.9966899 14 3.2666670.5889996 15 60 3.08125 0.4533412 16 2.62 0.408155 15 3.653333 0.765560315 3.38125 0.5472826 16 120 3.0875 0.4865589 16 3.126667 0.5076056 154.52 1.10122 15 3.65625 0.7101625 16

Cortisol blood plasma concentration: The mean cortisoi plasmaconcentrations over time after the administration of 8 IU-OT, 24 IU-OT,IV OT and Placebo (with error bars representing standard error of themean) are represented in Table 4 and FIG. 7(c), For the 4 (treatment)×6(time) LMM there was a significant main effect of treatment on cortisolblood plasma concentration [F(3,84,77)=4.82, p=0.004]. Fo pairwisecomparisons (q<0.05, revised critical value of p<0.017) revealedsignificantly increased cortisol concentration following IV-OT treatmentcompared to Placebo treatment (p=0.01) and 24 IU-OT (p<0.001), but not 8IU-OT. There was a significant main effect of time on cortisol bloodplasma concentration [F(5,90,07)=2,4, p=0.04], but no significantfollow-up pairwise comparisons were found. Finally, there was nosignificant treatment×time interaction [F(15,84,72)=0.421, p−0.969].

TABLE 4 8 IU-OT 24 IU-OT IV-OT Placebo Time Mean SEM N Mean SEM N MeanSEM N Mean SEM N −20 315.1875 32.40695 16 307.6249 45.70012 14 319.187536.74906 16 297.6667 33.23934 15 0 317.9375 28.85834 16 268.1429 36.812114 327.2667 41.42928 15 291.4 32.17849 15 10 286.3077 28.85605 13262.2857 31.79837 14 315.2 43.45257 15 268.875 29.88253 16 30 229.12520.52171 16 201.4286 24.05185 14 263.25 32.14855 16 223.8 19.33765 15 60208.625 18.48893 16 214.4 32.12739 15 253 29.17333 16 201.875 14.8985116 120 224.375 23.2185 16 239.4667 44.85382 15 263.875 31.46703 16239.9375 18.17449 16

In this study, it has been demonstrated that 8 IU-OT treatment reducesthe perception of anger in emotionally ambiguous facial stimuli withminimal systemic exposure, Importantly, the current findings are thefirst to suggest that a low dose of OT is more effective than a higherdose in modulating social cognition. Moreover, these results providebehavioral evidence that OT delivered intranasally using the deliverydevice of this study reaches the brain and influences social cognition,whereas peripherally administered OT, which similarly increased plasmaOT concentration, had no such effect.

This data highlights the subtle effect of OT on the processing ofemotionally ambiguous facial stimuli in relation to anger perception, asthere was no difference in the ratings of angry or happy faces. Whereasthe specific effects of OT in the emotionally ambiguous stimuli indicatethat OT only influences the emotional assessment of stimuli which arenon-abundant with overt cues, the lack of effects in the happy and angrystimuli could also be explained by the relatively low variability inratings of these stimuli, Notably, there were also no differences inratings of trust between the placebo condition and any of the OTconditions, While this may have been due to the explicit nature of the“trust” question [most research has used more nuanced economic tasks⁶⁴],this adds to mounting evidence that OT may not increase the perceptionof trustworthiness⁹⁶⁻⁹⁷.

The present delivery regime, which provides for efficacy with lower doseconcentrations, also has a particular advantage of enabling regulationof the balance of OT and AVP concentrations⁴⁹ via cross-reactivity withAVP receptors^(50,98-100). In addition, compared to higher doses, lowerdoses have been shown to increase peripheral levels of OT in salivas,attenuate cortisol stress responses⁶⁶, and increase eye gaze in patientswith Fragile X syndrome⁶⁷, Furthermore, a low dose of OT administeredshortly after birth has been shown to increase partner preference laterin life⁶⁸. Similarly, lower doses have been associated with strongerincreases in social recognition compared to higher doses⁶⁹⁻⁷⁰.

Much like OT, AVP receptors are located both centrally andperipherally⁷⁴⁻⁷⁵ and play an important role in social behavior andpsychopathology⁴⁹. It is postulated that this “off target” activity maycontribute to a non-linear dose-response and further highlights theimportance of establishing the dose regimen that optimizes therapeuticeffects¹⁰¹.

Importantly, the present dose-response data provides evidence to theoptimal dose for social cognition modulation, demonstrating that a lowerdose is more likely to modulate social cognition than a higher dose.Furthermore, patients with underlying deficits responsive to OT, mayrespond more robustly than healthy volunteers.

The present data on the perception of facial stimuli is generallyconsistent with results from past studies in humans, particularlynegatively valanced emotions⁸¹, as differences were only discovered onthe perception of anger in emotionally ambiguous faces. These resultsdocumenting specifically reduced negativity bias for emotionallyambiguous faces have important implications for disorders that arecharacterized by a negative bias towards social stimuli (e.g., socialanxiety disorder). Prior studies suggest that OT reduces bias towardsnegative information in clinically anxious⁸² and high trait anxiousindividuals⁸³; however, this is the first study to the presentinventors' knowledge to report data suggesting a reduction of negativitybias in healthy individuals,

Nasal Valve Dimension Analysis

Analysis was conducted using the R statistical package (version 3,1.1; RDevelopment Core Team, 2014) to examine the role of the cross-sectionalarea of the nasal valve, being the slit-like structure at the junctionbetween the anterior and posterior regions of each nasal cavity, onpharmacodynamics. A repeated-measures ANOVA was first conducted toinvestigate if the cross-sectional area of the nasal valve significantlyfluctuated from session-to-session (screening session and each treatmentsession). Additionally, as the cross-sectional area may differ accordingto an individuals' overall size and age, Pearson correlationcoefficients were calculated to assess the relationship between thesefactors at the time of screening.

The correlation between the response to angry ambiguous faces and themean cross-sectional area of the nasal valve was determined after 8IU-OT, 24 IO-OT, IV OT and Placebo treatments. In this study, asadministration was done to both the left and right nasal cavities, themean cross-sectional areas were determined for each of the left andright nasal cavities, and a mean cross-sectional area was determinedfrom the sum of these means for the left and right nasal cavities.

TABLE 5A Mean cross-sectional area of nasal valve for left nasal cavityMean SEM N Screening 0.664 0.056 16 8IU-OT 0.609 0.045 16 24IU-OT 0.6760.056 16 IV-OT 0.631 0.044 16 Placebo 0.746 0.091 16

TABLE 5B Mean cross-sectional area of nasal valve for right nasal cavityMean SEM N Screening 0.599 0.062 16 8IU-OT 0.619 0.058 16 24IU-OT 0.6140.064 16 IV OT 0.617 0.069 16 Placebo 0.561 0.052 16

TABLE 5C Mean cross-sectional area of nasal valve as determined from thesum of mean cross-sectional areas of nasal valves of left and rightnasal cavities Mean SEM N Screening 0.632 0.046 16 8IU-OT 0.614 0.035 1624IU-OT 0.645 0.042 16 IV OT 0.624 0.04 16 Placebo 0.654 0.049 16

Bayes Factors using the Jeffreys-Zellner-Siow method⁶⁰ were alsocalculated to assess the strength of evidence for the null andalternative hypotheses. This approach is especially useful indetermining if the data supports the null hypotheses (i.e., norelationship between two variables) over the alternative hypothesis(i.e., there is a relationship between two variables), as anon-significant p-value is unable to provide evidence for thenull-hypothesise. A Bayes value less than ⅓ provides substantialevidence for the null hypothesis, over 3 provides strong evidence forthe alternative hypothesis, and between ⅓ and 3 provides no strongsupport either way⁶³.

Confidence intervals for the difference between correlations for eachtreatment condition were calculated to compare the strength ofcorrelation to investigate whether the relationship between the meancross-sectional area of the nasal valve and anger ratings of ambiguousfaces is significantly greater than the relationships observed after theother treatments. As these variables are highly related due tomeasurements being taken from the same sampie⁶², the CIs were adjustedto account for overlap⁵⁸ using the Fisher Z transformation. Any CIinterval that includes zero would indicate that the null hypothesis ofno difference between the correlations could not be rejected.

The relationship between blood plasma and the mean cross-sectional areaof the nasal valve was also calculated, as represented in Table 5. Achange score between baseline OT and AVP and serum levels just beforethe social cognition assessment (˜40 minutes after treatment) wascalculated to explore the effect of the cross-sectional area of thenasal valve on OT, AVP and cortisol on systemic availability.

TABLE 6 The relationship between mean cross-sectional area of the nasalvalve and plasma concentration of oxytocin, vasopressin, and cortisol 8IU-OT 24 IU-OT IV-OT Placebo r 95% CI n p r 95% CI n p r 95% CI n p r95% CI n p Plasma OT .13 −.39, .59 15 .63 0.2 −.39, .68 12 0.51 −0.1−.56, .42 15 .73 .35  −.2, .73 14 .21 Plasma AVP .02 −.48, .51 15 .950.4 −.19, .78 12 0.17 .19 −.38, .65 13 .52 .38 −.19, .76 13 .18 Plasmacortisol .14 −.38, .6  15 .59 .29 −.31, .72 12 .34 −.22 −.64, .31 15 .42−.07 −.58, .47 13 .8

A repeated-measures ANOVA revealed no main effect of time for the meancross-sectional area of the nasal valve [F(1,99,29,86)==0.69, p=0.51;η_(p)=0.044], There was also no relationship between age [r=0.56, 95% CI(−0.45, 0.54), n=16, p=0.84] and BMI [r=−0.68, 95% CI (−0.55, 0.44),n=15, p=0.015] with the mean cross-sectional area of the nasal valve atthe time of screening.

The calculation of Pearson correlation coefficients revealed asignificant relationship between the anger ratings of neutral faces andthe mean cross-sectional area of the nasal valve after SIU-OT treatment[r=−0.61, 95% CI (−0.85, −0.14), n=15, p=0.015], with a correspondingBayes factor (B) of 3.62, representing substantial evidence that thesetwo variables are related. The relationship between angry ratings ofambiguous faces and the mean cross-sectional area of the nasal valvefollowing the 8 IU-OT treatment is represented in FIG. 8.

As represented in FIG. 8, there was no relationship between treatmentand anger ratings of neutral faces after 24 IU-OT treatment [r=−0.14,95% CI (−0.59, 0.38), n=16, p=0.6; B=0..22], IV-OT [r=0.11, 95% CI(−0.43, 0..59), n=15, p=0.7; B−0.21], or Placebo [r=0.04, 95% CI (−0.46,0.53), n=16, p=0.88; B=0.19] treatment, with all respective Bayesfactors indicative of substantial evidence that these variables are notrelated to each other.

A comparison of the correlation coefficients also revealed a significantdifference between the correlations of the 81 U-OT, and IV [r=0.72(−1.4, −0.2)] and Placebo [r=−0.65 (−1.1, −0.06)] treatments, but nosignificant difference in the correlation with 24 IU-OT treatment[r−0.42 (−0.97, 0.06)].

In addition, there was no relationship between the cross-sectional areaof the nasal valve and plasma concentration of OT, AVP, or cortisolafter any of the treatment conditions.

The present study evidences that the efficacy of OT on social cognitioncan be influenced by control of the cross-sectional area of the nasalvalve when intranasally administering a defined, lower-dosage of OT lessthan 24 IU. In one embodiment this control is obtained by the effectivepressure of the exhaled air flow and the structural effect of thenosepiece in opening the nasal valve.

fMRI Analysis

Conventional fMRI pre-processing of the fMRI data was performed usingindependent component analysis (ICA) and auto-classification using theFMRIB's ICA-based X-noiseifier (FIX) method in order to de-noise thefMRI data.

The individual components were grouped using a temporal concatenationapproach in MELODIC (Multivariate Exploratory Linear OptimisedDecomposition into Independent Components), fixed model order at 40components.

The component with strongest amygdala weighting (and also having strongmedial temporal lobe (MTL) and brain stem weighting) was thendetermined, here Independent Component #37 (IC0037).

Dual regression was then performed to estimate the spatial maps of theindividual components and the corresponding time courses, as representedin FIG. 9(a), which reflects one sample t-tests across all datasets(t>5) after dual regression.

Voxel-wise general linear model (GLM) testing was performed forevaluation of the main effect of the DT condition (F-test across theIU08-OT, IU24-OT, IV-OT and Placebo treatments) on the individualspatial maps within the canonical component (t>5) for IC0037. Thelargest clusters at voxel-wise p <0.01, uncorrected, were thenidentified. The two largest clusters showing the main effects of the OTcondition are localized within the left and right amygdale,respectively, as represented in FIG. 9(b).

Next, pairwise comparison between 8 IU-OT and Placebo treatmentsrevealed two clusters showing significantly (p<0.05, duster sizecorrected using permutation testing) increased connectivity in the 8IU-OT treatment as compared to Placebo in the left and right amygdala,respectively, as represented in FIG. 9(c). The mean connectivity valuefor each dataset in each of these four clusters was extracted andsubmitted to further analysis (here in MATLAB).

A repeated-measures ANOVA was performed. FIGS. 10(a) and (b) illustrateboxplots of the mean connectivity within the two clusters showingsignificant (p<0.01, uncorrected) main effects of the OT condition.FIGS. 11(a) and (b) illustrate boxpiots of the mean connectivity withinthe two clusters showing significantly (p<0.05, cluster size corrected)increased connectivity after the 8 IU-OT and Placebo treatments. Theconnectivity values are normalized (z scores) relative to each subject'smean value across conditions in order to ease comparison).

FIGS. 12(a) and (b) represent, by way of spaghetti plots, theconnectivity values in all conditions in each of the significantamygdala clusters obtained from the pairwise comparison, as illustratedin FIG. 9(c), for each individual,

As expected, repeated-measures ANOVA revealed significant main effectsof condition in both clusters (p=0.032 and p=0.0039), Boxplots suggestthat main effects of OT condition are driven by IU08-OT vs Placebo,indicating increased amygdala connectivity in the IU08-OT treatment,which is also supported by post-hoc pairwise comparisons (t=−2.54,p=0.016, and t=−2.24, p=0.033).

The amygdala is a key brain region for emotion regulation⁸⁶, playing animportant role in processing incoming social stimull⁸⁷. Indeed,converging neuroimaging evidence suggests the amygdala is an importanttarget of OT administration. For instance, a single administration ofintranasal OT has been reported to both decrease⁸⁸⁻⁸⁹ and increase⁹⁰⁻⁹¹amygdala activity when viewing a range of emotional stimuli. While theseearly studies measured neuronal recruitment during the presentation ofstimuli, recent work has begun to explore brain activity at rest. It isreported that the amygdala is a key constituent of a larger “socialbrain network” that displays increased blood flow after OTadministration⁹². Similarly, data indicates that OT administrationincreases connectivity between the amygdala and the rostral medialfrontal cortex⁹³.

The present study is the first to examine resting state connectivityafter OT administration of different doses (8 IU and 24 IU) andtreatment modalities (intranasal vs. intravenous). The data suggeststhat a low dose of OT delivered intranasally (but not intravenously)modulates amygdala connectivity, which is consistent with nose-to-braindelivery. Increased amygdala connectivity may facilitate the increasedsalience of social stimuli, which is suggested to underpin the observedeffects of OT on social cognition and behaviorw. These results may alsohave implications for the treatment of psychiatric disorderscharacterized by social impairment, which are also reported to haveabnormal coupling between the amygdala and other brain regions (e.g.,schizophrenia)⁹⁴. Moreover, the data also adds to our understanding ofhow different OT doses and administration modalities influence neuronalrecruitment at rest.

In summary, the present study presents new insights in relation to animproved method of deep intranasal OT delivery, and shows that greaterpharmacodynamic activity can be shown specifically using the presentdelivery regime of OT as compared to IV delivery producing similarsystemic exposure, suggesting that direct nose-to-brain activity isbeing achieved. This data also provides preliminary evidence that theselection of intranasal OT dose based on precedence, rather thanexperimental evidence, may be misguided; the current study indicatingthat a lower dose (8 IU) can offer greater efficacy than a higher dose(24 IU) when suitably administered.

MRI and Pupilometry Analysis

FreeSurfer (http://surfer.nmr.mgh.harvard.edu) was used for of theT1-weighted data, including surface reconstruction and full brainsegmentation¹²³ to obtain precise brain extracted volumes forco-registration of the fMRI data. FRRIB Software Library (FSL;http://fsl.fmrib.ox.ac.uk/fsl/fsiwiki/¹²⁴) was used to process fMRIdata, The first five volumes were discarded. Pre-processing of fMRI datawas conducted using FMRIB's Expert Analysis Tool (FEAT) version 6.0¹²⁸,This included motion correction using MCFLIRT¹²⁴, spatial smoothing bymeans of SUSAN¹²⁵ using a Gaussian kernel of FWHM of 7 mm, and atemporal high pass filter of 100 s. Single session independent componentanalysis (ICA) was performed using Multivariate Exploratory LinearOptimized Decomposition into Independent Components (MELODIC ICA¹²⁶) inorder to perform automated denoising (see below). FMR/B's Linear andnon-linear Image Registration Tools (FLIRT¹²⁴) optimized using BoundaryBased Registration (BBR¹²⁷) was used to align each participant's fMRIdata to a standard space (MNI-152) with the T1-weighted volume as anintermediate.

Individual level general linear models (GLM) were fitted using FILM(FMRIB's Improved Linear Model)¹²⁷⁻¹²⁸ modeling the facial stimuli(happy/angry/ambiguous faces) and geometrical shape as events with theinterspersed fixation trials as implicit baselines. Q1 and Q2 weremodeled as one regressor across the different facial stimuli and shapes.Next, the average amygdala contrast-parameter estimates (COPE) wereextracted from left and right amygdala masks based on the Harvard-Oxfordanatomical atlas provided with FSL and submitted the values tohigher-level linear mixed models in SPSS to test for main effects ofcondition and treatment (see below). Pupilometry data was pre-processedusing a custom made MATLAB-script. Raw data were converted intodiameters, with physiologically unlikely pupil sizes (<2 mm or >9 mm)excluded from the data to remove noise (e.g., eye blinks). Each timeseries was split into trials with the average pupil diameter from eachstimuli condition calculated. Finally, the first 8 seconds across all 20trials for each condition were averaged to generate mean overall pupildiameters.

Statistical analysis was conducted using IBM SPSS Statistics version 22(IBM, Armonk, N.Y.) to examine the impact of treatment on amygdalaactivity. As described above, a linear mixed-model (LMM) approach wasadopted for the analysis of amygdala activity. All models were fittedusing an unstructured matrix. Experimental treatment was both a fixedand repeated effect in the LMM testing the impact of treatment onamygdala activity, The same LMM approach was used to examine differencesin mean pupil diameter, COPE values for contrasts of both left and rightamygdala activity between angry faces and shapes, happy faces andshapes, and happy faces and angry faces, Standardized residuals aftermodel fitting were examined for outliers. Z-scores above 2.58 or below−2.58 were removed from the analysis. Outliers beyond these thresholdswere removed from the amygdala activation datasets (1 value from theright amygdala data during the presentation of angry, happy and,ambiguous, and shape stimuli, respectively; 1 value from left amygdalaanger and happy data, respectively; and 2 values from the left amygdalaambiguous and shape data, respectively). For any significant maineffects (p<0.05), post-hoc tests were performed to compare eachtreatment condition with the adjustment of critical p values to correctfor multiple comparisons using a 5% false discovery rate (FDR)⁵⁹. Therelationships between amygdala activation and; mean pupil dilation,behavioral ratings, and nasal physiology were also assessed. Finally,Bayes Factors using the Jeffreys-Zeliner-Siovv prior⁶⁰ were calculatedto examine the strength of evidence for both the null and alternativehypotheses.

LMM revealed a significant main effect of treatment on right amygdalaactivity during the presentation of angry faces [F(3,15.1)=4.54,p=0.019; FIGS. 13(a) and (b)]. Follow-up pairwise comparisons (q=0.05,revised critical value of p<0.008) indicated that right amygdalaactivation was significantly reduced in the 8 IU-OT treatment conditionin comparison to placebo (p=0.002). There was a main effect of treatmenton right amygdala activity in response to the presentation of happyfaces [F(3,15)=3.44, p=0.04], with posthoc comparisons indicating thereduction after 8 IU-OT compared to placebo was on the border of the FORsignificance threshold (p=0.01; q=0.05, revised critical value ofp<0.008). There was a main effect of treatment, on the border ofsignificance, for right amygdala activity during the presentation ofambiguous faces [F(3,14.6)=3.15, p=0.057], Exploratory posthoc analysesrevealed the reduction of right amygdala activity in the 8 IU OTcondition compared to the placebo condition was on the border of the FORcorrected significance threshold (p=0.01; q=0.05, revised critical valueof p<0.008). There was also a main effect of treatment and geometricshapes [F(3,15)=3.56, p=0.04], however, post hoc analyses revealed nosignificant differences after FLA corrected thresholds. There was a maineffect for the happy faces>angry faces contrast for the right amygdala[F(3,14.7)=4.46, p=0.02] but no posthoc comparisons survived FORcorrected thresholds. With regard to left amygdala activity, a LMMrevealed no main effect of condition during the presentation of angryfaces [F(3,15,1)=1.28, p=0.32], ambiguous faces [F(3,13.6)=1.14,p=0.37], happy faces [F(3,14)=2.14, p=0.14], or geometric shapes[F(3,14,4)=1.87, p=0.18]. There was a main effect for the happyfaces>angry faces contrast on left amygdala activity [F(3,14.7) =4.79,p=0.02], but no posthoc comparisons survived FDR corrected thresholds.There were no main effects of treatment for any of the emotion>shapeCOPE value contrasts, as represented in Table 7.

TABLE 7 COPE values for amygdala activity Linear mixed model main effect8 IU-OT 24 IU-OT IV-OT Placebo df F P Right amygdala Angry faces >shapes .36 (.5) −.13 (.01)  −.12 (.01) −.12 (.01)  3, 15  0.43 0.74Happy faces > shapes −.61 (.17) .02 (.27) −.24 (.22) .26 (.32) 3, 14.70.45 0.72 Ambiguous faces > shapes −.09 (.19) −.22 (.33)   .16 (.23) .14(.24) 3, 15.1 0.48 0.7 Left amygdala Angry faces > shapes  −.19 (.004).54 (.49) −.17 (.01) −.18 (.01)  3, 15  2.09 0.14 Happy faces > shapes−.05 (.19) 1.3 (.23) −.34 (.2)  .11 (.36) 3, 14.3 2.44 0.11 Ambiguousfaces > shapes −.11 (.19) .02 (.37)  .02 (.24) .02 (.22) 3, 14.8 0.110.95 Note. Values represent z-score estimated marginal means withstandard errors in parenthesis.

There was no significant main effect of treatment on mean pupil diameterwhile processing angry [F(3,15)=0.57, p=0.64], happy [F(3,15)=0.62,p=0.62], or emotionally ambiguous faces [F(3,15)=1.33, p=0.3]. However,there was a significant relationship between right amygdala activationand mean pupil diameter during the presentation of, angry (p=0.02; FIG.14(a)), ambiguous (p<0.001; FIG. 14(b)), and happy (p=0.01; FIG. 14(c))faces after 8 IU-OT treatment, as represented in Table 8. All thecorresponding Bayes factors (B) were greater than 3, providingsubstantial evidencelm that these two variables are related. There wereno significant relationships after the other treatments (All p's>0.05),and all B's were less than 0.33, providing substantial evidence thatnone of these variables were related. Finally, there were no significantrelationships between intensity of anger ratings and right amygdalaactivity after any of the treatments, as represented in Table 9, orbetween nasal valve dimensions and right amygdala activation in afterany of the treatments, as represented in Table 10. As describedhereinabove, there was no difference in nasal valve dimensions beforeeach treatment administration [F(9, 108)=0.41, p=0.93). The frequency ofadverse events (e.g., brief dizziness) reported was equivalent betweentreatment groups (8 IU-OT, three reports; 24 IU-OT, two reports, IV OT,three reports, placebo, two reports).

TABLE 8 Relationship between pupil diameter and amygdala activationafter each treatment 8 IU-OT ^(a) 24 IU-OT ^(b) IV-OT ^(b) Placebo ^(b)r (95% CI) p B r (95% CI) p B r (95% CI) p B r (95% CI) p B Pupildiameter - .61 (.14, .86) .02 3.53 .09 (−.42, .56) 0.73 .2 −.22 (−.65,.31) 0.4 .26 .24 (−.29, .66) .38 .28 Angry faces Pupil diameter - .79(.46, .93) <.001 82.7 −.04 (−.53, .46)  0.89 .19 −.11 (−.57, .41) .68.21 .07 (−.44, .55) .81 .2 Ambiguous faces Pupil diameter - .63 (.17,.86) .01 4.53 .02 (−.48, .51) 0.95 .19 −.18 (−.62, .35) .5 .24 .22(−.31, .65) .42 .26 Happy faces Note. ^(a) N = 15, ^(b) N = 16; B =Bayes Factor

TABLE 9 Relationship between anger ratings and right amygdala activationafter each treatment 8 IU-OT 24 IU-OT ^(c) IV-OT Placebo ^(c) r (95% CI)p B r (95% CI) p B r (95% CI) p B r (95% CI) p B Angry faces  .07 (−.48,.58) ^(a) .8 .21  .05 (−.46, .53) 0.87 .19 −.01 (−.52, .5) ^(b)  .97 .19.29 (−.24, .67) .28 .34 Happy faces .14 (−.4, .61) ^(b) .62 .22 −.42(−.76, .1)  0.11 .7 −.47 (−.79, .06) ^(b) .07 .93 .21 (−.32, .64) .44.26 Ambiguous faces −.03 (−.55, .51) ^(a ) .92 .2 −.44 (−.77, .07) 0.09.81 −.19 (−.63, .34) ^(c) .51 .24 −.09 (−.56, .42)  .74 .2 Note. ^(a) N= 14, ^(b) N = 15, ^(c) N = 16; B = Bayes Factor.

TABLE 10 Relationship between nasal valve dimensions and right amygdalaactivation after each treatment 8 IU-OT ^(a) 24 IU-OT ^(b) IV-OT ^(b)Placebo ^(b) r (95% CI) p B r (95% CI) p B r (95% CI) p B r (95% CI) p BAngry faces −.03 (−.53, .49) .92 .2 −.07 (−.55, .44) .81 .2 .21 (−.32,.64) .44 .27 −.11 (−.57, .41) .68 .21 Happy faces  .03 (−.49, .53) .91.2 −.11 (−.57, .41) .69 .21 .17 (−.36, .61) .54 .23 −.17 (−.61, .36) .54.23 Ambiguous faces −.15 (−.62, .39) .62 .22 −.03 (−.52, .47) .91 .19−.12 (−.58, .4)  .65 .21 −.17 (−.61, .36) .54 .23 Note. ^(a) N = 15,^(b) N = 16; B = Bayes Factor.

In this study, 8 IU-OT treatment is shown to reduce amygdala activity incomparison to placebo. These findings are the first to report directcomparison of nose-to-brain and systemic delivery of OT, and indicatethat OT delivery via nose-to-brain pathways—but not peripherallydelivered OT producing similar blood levels—replicates awell-characterized finding of reduced right amygdala activation inresponse to emotional stimuli after OT treatment^(88,114-115).

Significantly, this data is consistent with the findings as discussedabove that OT deiivereci by the inventive device modulates theperception of anger in facial stimuli and with animal models thatassociated a lower OT dose with stronger increases in socialrecognition⁶⁹⁻⁷⁰, which is pertinent given the important role of theamygdala in social cognition and behavior.

These effects may not be specific to negatively-valenced social stimulias the main effects of treatment on right amygdala activity during thepresentation of happy and ambiguous faces were significant and on theborder of significance, respectively. Subsequent posthoc comparisonsbetween the 8 IU-OT treatment and placebo were on the border ofstatistical significance. The observed reductions in right amygdalaactivity during the presentation of both positively and negativelyvalanced stimuli after OT treatment are consistent with the hypothesisthat OT increases approach-related behaviours^(114,118).

Secondary analysis revealed a significant association between rightamygdala activity and mean pupil diameter during the processing ofangry, ambiguous, and happy facial stimuli after 8 IU-OT administration.While a main effect of treatment on pupil diameter not was found, thedata is indicative of the amygdala modulating cognitive resources tofacial stimuli, regardless of valence, after 8 IU-OT treatment.

The amygdala is a site of large number of oxytocin receptors¹³¹⁻¹³².These receptors have been shown to operate by inhibiting amygdalaactivity via the increase of GABAergic interneuron activityl³³⁻¹³⁴. Theobserved decrease in amygdala activity after OT administration using theinventive device is consistent with nose-to-brain molecule transport viaolfactory and trigeminal nerve fiber pathways¹³⁵, Outputs to theamygdala via the olfactory buibs¹³⁶⁻¹³⁸ or transport through brainextracellular fluid¹³⁹ from olfactory bulb and brainstem delivery sitesmay facilitate these reductions in amygdala activity via a localGABAergic circuit after intranasal delivery, Irrespective of howendogenous OT precisely affects amygdala activity, by having aperipheral comparator this study demonstrates that nose-to-brainpathways produce effects not observed with comparable levels of purelysystemic exposure, suggesting facilitated entry to the brain.

The dose-response data reported here suggest that a low dose of OTdelivered using the inventive device is sufficient to modulate amygdalaactivity. Patients with underlying deficits responsive to OT may respondmore robustly than healthy volunteers.

There are a number of reasons that may explain why an effect was foundwith the 8 IU-OT dose but not the 24 IU-OT, These include crossreactivity with vasopressin receptors⁴⁹ and the possibility that an 8IU-OT dose delivered with the inventive device is better able to reachthe regions in the nose where direct nose-to-brain transport can occur,

Significantly, no evidence was found that 1 IU-OT of peripherallyadministered OT influences amygdala activity. Although there isconflicting evidence on whether peripheral OT can cross the BBE¹⁴⁰⁻¹⁴¹,our study suggests that even if OT does travel across this barrier insmall amounts, this quantity is not large enough to modulate amygdalaactivity compared to placebo. Individual differences and context caninfluence the response to OT administration'⁶, thus a strength of thisstudy was the use of a within-subjects design to examine amygdalaactivity, By adopting this experimental design, any individualdifferences due to variation in the endogenous oxytocin system¹⁴²⁻¹⁴³are minimized.

In summary, the present study shows surprisingly that a low dose of OTintranasally delivered with the described delivery method modulatesamygdala activity, and this result provides additional evidence tosuggest a lower intranasal CT dose may better facilitate the modulationof social cognition and behavior and that peripheral actions of CT donot appear to have any significant neural coronariae.

REFERENCES

-   1, Guastelia A J, MacLeod C (2012): A critic& review of the    influence of oxytocin nasal spray on social cognition in humans:    Evidence and future directions, Horm Behav, 61:410-418,-   2. Meyer-Lindenberg A, Domes G, Kirsch P, Heinrichs M, Oxytocin and    vasopressin in the human brain: social neuropeptides for    translational medicine, Nat Rev Neurosci 2011; 12: 524-538,-   Striepens N, Kendrick K M, Maier W, Hurlemann R. Prosoclai effects    of oxytocin and clinical evidence for its therapeutic potential.    Front Neuroendocrinol 2011; 32:426-450.-   Bartz J A, Zaki J, Bolger N, Hollander E, Ludwig N N, Kolevzon A, at    al (2010): Oxytocin Selectively Improves Empathic Accuracy, Psychol    Sci. 21:1426-1428.-   5. Hurlemann R, Patin A, Onur O A, Cohen M X, Baumgartner T, Metzler    S et al. Oxytocin enhances amygdala-dependent, socially reinforced    learning and emotional empathy in humans. J Neurosci 2010; 30:    4999-5007,-   Kosfeid M, Heinrichs M, Zak P J, Fischbacher U, Fehr E. Oxytocin    increases trust in humans. Nature 2005; 435: 673-676.-   Shalvi S, De Dreu C K, Oxytocin promotes group-serving dishonesty.    Proc Natl Acad Sci USA 2014; 111: 5503-5507.-   8, Van Ijzendoorn M H, Bakermans-Kranenburg M J. A sniff of trust:    meta-analysis of the effects of intranasal oxytocin administration    on face recognition, trust to ingroup, and trust to out-group.    Psychoneuroendocrinology 2012; 37: 438-443.-   Guastella A J, Mitchell P B, Dadds M R (2008): Oxytocin increases    gaze to the eye region of human faces, Biol Psychiatry. 63:3-5.-   10 Domes G, Heinrichs M, Michel A, Berger C, Herpertz S C (2007):    Oxytocin Improves “Mind-Reading” in Humans. Biol Psychiatry.    61:731-733,-   11. Guastella A J, Einfeld S L, Gray K M, Rinehart N J, Tonge B I,    Lambert T J, et al (2010): Intranasal Oxytocin Improves Emotion    Recognition for Youth with Autism Spectrum Disorders. Biol    Psychiatry. 67:692-694.-   12. Medi M E, Young L J (2012): The oxytocin system in drug    discovery for autism; Animal models and novel therapeutic    strategies, Horm Behav. 61:340350-   13. MacDonald K, Feifel D (2012); Oxytocin in schizophrenia: a    review of evidence for its therapeutic effects. Acta    Neuropsychiatrica, 24:130-146.-   14. Dadds M R, MacDonald E, Cauchi A, Williams K, Levy F, Brennan J    (2014): Nasal oxytocin for social deficits in childhood autism: A    randomized controlled trial. J Autism Dev Disord. 44:521-531.-   15. Guastella A J, dickie I B, McGuinness M M, Otis M, Woods E A,    Risinger H M, et al (2013): Recommendations for the standardisation    of oxytocin nasal administration and guidelines for its reporting in    human research, Psychoneuroendocrinology. 38:612-625.-   16. Bartz J A, Zaki J, Bolger N, Ochsner K N (2011): Social effects    of oxytocin in humans; context and person matter. Trends in    cognitive sciences, 15;301-309.-   17. MacDonald K, Feifei D (2013): Helping oxytocin deliver:    considerations in the development of oxytocin-based therapeutics for    brain disorders, Front Neurosci.-   18. Quintana D S, Aivares G A, Hickie I B, Guastella A J (2015): Do    delivery routes of intranasally administered oxytocin account for    observed effects on social cognition and behavior? A two-level    model. Neurosci Biobehav Rev. 49:182-192.-   19. Landgraf R, Neumann I D. Vasopressin and oxytocin release within    the brain: a dynamic concept of multiple and variable modes of    neuropeptide communication, Front Neuroendocrinol 2004; 25: 150-176.-   20. Iliff J J, Wang M, Liao Y, Plogg B A, Peng W, Gundersen G A et    al. A paravascular pathway facilitates CSF flow through the brain    parenchyma and the clearance of interstitial solutes, including    amyloid β. Sci Transi Med 2012; 4:147ra111-147ra111.-   21. Dhuria S V, Hanson L R, Frey W H I I. Intranasal delivery to the    central nervous system: Mechanisms and experimental considerations.    J Pharm Sci 2009; 99:1654-1673.-   22. Ermisch A, Barth T, Rutile H, Skopkova J, Hrbas P, Landgraf R.    On the blood-brain barrier to peptides: accumulation of labelled    vasopressin, DesGlyNH2-vasopressin and oxytocin by brain regions.    Endocrinol Exp 1985; 19: 29-37.-   23. Djupesland P G, Messina J C, Mahmoud R A (2014): The nasal    approach to delivering treatment for brain diseases: an anatomic,    physiologic, and delivery technology overview. Therapeutic delivery.    5:709-733.-   24. Cole P (2003): The four components of the nasal valve, Am J    Rhinol. 17:107410.-   25. Aggarwal R, Cardozo A, Homer J. The assessment of topical nasal    drug distribution. Clin Otoiaryngol Awed Sci 2004; 29: 201-205.-   26. Djupesland P G, Skretting A, Winderen M, Holand T (2006): Breath    Actuated Device Improves Deliver^(,)/ to Target Sites Beyond the    Nasal Valve. The Laryngoscope. 116:466-472.-   27. Djupesiand P G, Messina J C, Mahmoud R A. Breath powered nasal    delivery: a new route to rapid headache relief. Headache 2013; 53:    72-84.-   28. Eccles R. Nasal airflow in health and disease. Acta Otoiarynool    2000; 120: 580-595.-   29. Merkus P, Ebbers F A, huller B, Fokkens W J. Influence of    anatomy and head position on intranasal drug deposition. Eur Arch    Otorninolaryngol 2006; 263:827-832.-   30. Djupesland P G, Mahmoud R A, Messina J C (2013): Accessing the    brain: the nose may know the way. Journal of Cerebral Blood Flow &    Metabolism. 33:793-794.-   31. Djupesland P G, Skretting A (2012): Nasal Deposition and    Clearance in Man; Comparison of a Bidirectional Powder Device and a    Traditional Liquid Spray Pump. Journal of Aerosol Medicine and    Pulmonary Drug Delivery. 25:280-289.-   32. Djupesiand P G (2012): Nasal drug delivery devices;    characteristics and performance in a clinical perspective—a review.    Drug Delivery and Translational Research 2012; 3:42-62.-   33. Hollander E, Novotny S, Hanratty M, Yaffe R, DeCada C M,    Aronowitz B R, et al (2003): Oxytocin Infusion Reduces Repetitive    Behaviors in Adults with Autistic and Asperger&apos;s Disorders,    Neuropsychopharmacology, 28;193-198.-   34. Hollander E, Bartz J, Chaplin W, Phillips A, Sumner J, Soorya L,    et al (2007): Oxytocin Increases Retention of Social Cognition in    Autism, Biol Psychiatry, 61:498-503.-   35. Striepens N, Kendrick K M, Hanking V, Landgraf R, Wüllner U,    Maier W et al. Elevated cerebrospinal fluid and blood concentrations    of oxytocin following its intranasal administration in humans, Sci    Rep 2013; 3: 3440.-   36. MacDonald E, Dadds M R, Brennan J L, Williams K, Levy F, Cauchi    A J (2011): A review of safety, side-effects and subjective    reactions to intranasal oxytocin in human research,    Psychoneuroendocrinology. 36:1114-1126.-   37. Bakermans-Kranenburg M, Van Ijzendoorn M (2013): Sniffing around    oxytocin: review and meta-analyses of trials in healthy and clinical    groups with implications for pharmacotherapy, Translational    psychiatry, 3:e258.-   38. de Oliveira D C, Zuardi A W, Graeff F C, Queiroz R H, Crippa J A    (2012): Anxiolytic-like effect of oxytocin in the simulated public    speaking test. J Psychopharmacol (Oxf). 26;497-504.-   39. Butwick A, Coleman L, Cohen S, Riley E, Carvalho B (2010):    Minimum effective bolus dose of oxytocin during elective Caesarean    delivery, Br Anaesth, 104;338-343.-   40. Rauit J-L, Carter C S, Garner J P, Marchant-Forde J N, Richert S    T, Lay Jr D C (2013): Repeated intranasal oxytocin administration in    early life dysregulates the HPA axis and alters social behavior.    Physiol Behav. 112:40-48.-   41. Gimpi G, Fahrenholz F (2001): The oxytocin receptor system:    structure, function, and regulation, Physiol Rev. 81;629-683.-   42. Mayer-Hubner B (1996): Pseudotumour cerebri from intranasal    oxytocin and excessive fluid intake. The Lancet. 347:623-623.-   43. Karat M, Heinrichs M, Schwarzwald R, Domes G. Oxytocin    attenuates neural reactivity to masked threat cues from the eyes.    Neuropsychopharmacology 2015; 40: 287-295.-   44. Evans S, Shergill S S, Averbeck B B. Oxytocin decreases aversion    to angry faces in an associative learning task.    Neuropsychopharmacology 2010; 35.2502-2509.-   45. Domes G, Steiner A, Porges S W, Heinrichs M. Oxytocin    differentially modulates eye gaze to naturalistic social signals of    happiness and anger. Psychoneuroendocrinology 2013; 38: 1198-1202.-   46. Jess: S, Morlog D, Ross S, Pell M D, Pasternak S H, Mitchell D G    et al. The effects of oxytocin on social cognition and behaviour in    frontotemporal dementia, Brain 2011; 134: 2493-2501.-   47. Bertsch K, Gamer M, Schmidt B, Schmidinger I, Walther S, Kästel    T et ad. Oxytocin and reduction of social threat hypersensitivity in    women with borderline personality disorder. Am J Psychiatry 2013;    170: 1169-4177.-   48. MacDonald K, Feifel D. Oxytocincs role in anxiety: a critical    appraisal, Brain Res 2014; 1580: 22-56.-   49. Neumann I D, Landgraf R (2012): Balance of brain oxytocin and    vasopressin: implications for anxiety, depression, and social    behaviors. Trends Neurosci, 35:649-659.-   50. Legros J,Chiodera P, Geenen V, Smitz S, Frenckell Rv (1984):    Dose-Response Relationship between Plasma Oxytocin and Cortisol and    Adrenocorticotropin Concentrations during Oxytocin Infusion in    Normal Men*. The Journal of Clinical Endocrinology & Metabolism    58:105-109.-   51. Neumann I D. Involvement of the brain oxytocin system in stress    coping: interactions with the hypothaiamo-pituitary-adrenal axis.    Frog Brain Res 2002; 139; 147-162.-   52. Wechsler D (1999): Weschsler Abbreviated Scale of Intelligence.    San Antonio, Tex.: Psychological Corporation.

53. Lecrubier Y, Sheehan D, Weiner E, Amorim P, Bonora I, HarnettSheehan K, et al (1997): The Mini International NeuropsychiatricInterview (MINI). A short diagnostic structured interview: reliabilityand validity according to the CIDI. Eur Psychiatry. 12:224-231.

-   54. Spielberger C D (1983): Manual for the State-Trait Anxiety    Inventory STAI (form Y)(“self-evaluation questionnaire”).-   55. Leknes S, Wessberg J, Eilingsen D M, Chelnokova O, Olausson H,    Laeng B (2012): Oxytocin enhances pupil dilation and sensitivity to    ‘hidden’ emotional expressions. Soc Cogn Affect Neurosci. 8:741-749.-   56. Lundgvlst D, Flykt A, Ohman A (1998): The Karolinska directed    emotional faces (KDEF), CD ROM from Department of Clinical    Neuroscience, Psychology section, Karolinska Institutet. 91-630.-   57. McCullough M E, Churchiand P S, Mendez A J. Problems with    measuring peripheral oxytocin: can the data on oxytocin and human    behavior be trusted? NeurosciBiobehav Rev 2013; 37: 1485-4492.-   58. Hamer R, Simpson P (2009): Last observation carried forward    versus mixed models in the analysis of psychiatric clinical trials.    Am J Psychiatry, 166:639-641.-   59. Benjamini Y, Hochberg Y (1995): Controlling the false discovery    rate: a practical and powerful approach to multiple testing. Journal    of the Royal Statistical Society Series B (Methociological).    289-300.-   60. Wetzeis R, Wagenmakers, E-J (2012). A default Bayesian    hypothesis test for correlations and partial correlations. Psychon.    Bull. Rev. 19, 1057-4064.-   61. Zou G Y (2007). Toward using confidence intervals to compare    correlations. Psychol. Methods 12, 399.-   62. Raghunathan T, Rosenthal R, Rubin, D B (1996). Comparing    correlated but nonoverlapping correlations, Psychol. Methods 1, 178.-   63. Dienes Z (2014). Using Bayes to get the most out of    non-significant results. Front Psychol 2014; 5:1-17.-   64. Theodoridou A, Rowe A C, Penton-Voak I S, Rogers P J (2009):    Oxytocin and social perception: oxytocin increases perceived facial    trustworthiness and attractiveness, Horm Behav. 56:128-132.-   65. Van Ijzendoorn M H, Bhandari R, Van der Veen R, Greweri K M,    Bakermans-Kranenburg M J (2012): Elevated salivary levels of    oxytocin persist more than 7 h after intranasal administration,    Front Neurosci. 6.-   66. Cardoso C, Ellenbogen M A, Orlando M A, Bacon S L, Joober R    (2013):

Intranasai oxytocin attenuates the cortisol response to physical stress:a dose-response study. Psychoneuroendocrinology. 38:399-407.

-   67. Hall S S, Lightbody A A, McCarthy B E, Parker K J, Reiss A L    (2012): Effects of intranasal oxytocin on social anxiety in males    with fragile X syndrome. Psychoneuroendocrinology, 37:509-518.-   68. Bales K L, Perkeybile A M, Conley O G, Lee M H, Guoynes C D,    Downing G M, at al (2013): Chronic intranasal oxytocin causes    long-term impairments in partner preference formation in male    prairie voles. Biol Psychiatry, 74:180-188.-   69. Benelli A, Bertalini A, Poggioli R, Menozzi B, Basaglia R,    Arletti R (1995): Polyniodal dose-response curve for oxytocin in the    social recognition test, Neuropeptides. 28:251-255.-   70. Popik P, Vetulani J, Van Rae J M (1992): Low doses of oxytocin    facilitate sodal recognition in rats. Psychopharmacology (Berl).    106:71-74.-   71. Bloom D E, Cafiero E, Jané-Llopis E, Abrahams-Gessel S, Bloom L    R, Fathirna S at al. The Global Economic Burden of Noncommunicable    Diseases: Program on the Global Demography of Aging, 2012.-   72. Miller G. Is pharma running out of brainy ideas. Science 2010;    329: 502-504.-   73. Abbott A. Novartis to shut brain research facility, Nature 2011;    480: 161-462.-   74. Frank E, Landgraf R (2008): The vasopressin system—from    antidiuresis to psychopathology. Eur J Pharmacol. 583:226-242.-   75. Li C, Wang W, Summer S N, Westfall T D, Brooks D P, Falk S, et    al (2008): Molecular mechanisms of antidiuretic effect of oxytocin.    J Am Soc Nephrol. 19:225-232.-   76. Weisman O, Schneiderman I, Zagoory-Sharon O, Feldman R, Salivary    vasopressin increases following intranasal oxytocin administration.    Peptides 2013; 40: 99-103.-   77. Burri A, Heinrichs M, Schedlowski M, Kruger T H. The acute    effects of intranasal oxytocin administration on endocrine and    sexual function in males, Psychoneuroendocrinology 2008; 33:    591-600.-   78. Gossen A, Hahn A, Westphal L, Prinz S, Schultz R, Grinder G et    al, Oxytocin plasma concentrations after single intranasal oxytocin    administration—A study in healthy men. Neuropeptides 2012; 46:    211-215,-   79. Charlton S, Davis S, Ilium L. Nasal administration of an    angiotensin antagonist in the rat model: effect of bioadhesive    formulations on the distribution of drugs to the systemic and    central nervous systems. Int J Pharm 2007; 338: 94-103.-   80. Dale O, Nilsen T, Loftsson T, Tonnesen H H, Klepstad P, Kaasa S    et al. Intranasal rhidazolam: a comparison of two delivery devices    in human volunteers. J Pharm Pharmacol 2006; 58: 1311.1318.-   81. Shahrestani S, Kemp A H, Guastella A J (2013): The impact of a    single administration of intranasal oxytocin on the recognition of    basic emotions in humans: a meta-analysis. Neuropsychopharmacology.    38:1929-1936.-   82. Guestella A J, Howard A L, Dadds M R, Mitchell P, Carson D S    (2009): A randomized controlled trial of intranasal oxytocin as an    adjunct to exposure therapy for social anxiety disorder.    Psychoneuroendocrinology, 34:917-923.-   83. Elford R C, Nathan P J, Auyeung B, Mogg K, Bradley B P, Sule A    et al. Effects of oxytocin on attention to emotional faces in    healthy volunteers and highly socially anxious males. Int J    Neuropsychopharmacol 2014; 18: 1-11.-   84. Alvares G A, Chen N T M, Balleine B W, Hickie I B, Guasteila A J    (2012): Oxytocin selectively moderates negative cognitive appraisals    in high trait anxious males. Psychoneuroendocrinology. 37:2022-2031,-   85. Fisher R A (1935). The design of experiments.-   86. LeDoux J E (2001). Emotion circuits in the brain. The Science of    Mental Health: Fear and anxiety 259.-   87. Seeley W W, Merron V, Schatzberg A F, Keller J, Glover G H,    Kenna H, Reiss A L, Greicius M D (2007), Dissociable intrinsic    connectivity networks for salience processing and executive control.    The Journal of neuroscience 27, 2349-2356.-   88. Domes G, Heinrichs M, Glascher J, Büchel C, Braus D F, Herpertz    S C (2007). Oxytocin attenuates amygdala responses to emotional    faces regardless of valence. Biol. Psychiatry 62, 1187-4190.-   89. Kirsch P, Esslinger C, Chen Q, Mier D, Lis 5, Siddhanti S,    Gruppe H, Mattay V S, Gallhofer B, Meyer-Lindenberg A (2005),    Oxytocin modulates neural circuitry for social cognition and fear in    humans, The Journal of neuroscience 25, 11489-11493.-   90. Domes G, Lischke A, Berger C, Grossmann A, ilauenstein K,    Heinrichs M, Herpertz SC (2010). Effects of intranasal oxytocin on    emotional face processing in women. Psychoneuroendocrinology 35,    83-93.-   91. Gamer M, Zurowski B, Buchel C (2010). Different amygdaia    subregions mediate vaience-related and attentional effects of    oxytocin in humans. PNAS 107, 9400-9405.-   92. Paloyelis Y, Doyle O M, Zelaya F O, Maltezos S, Williams S C,    Fotopoulou A, Howard M A (2014). A Spatiotemporal Profile of in vivo    Cerebral Blood Plea Changes Following Intranasal Oxytocin in Humans.    Biol, Psychiatry.-   93. Sripada C S, Phan K L, Labuschagne I, Welsh R, Nathan P. J, Wood    A G, (2013). Oxytocin enhances resting-state connectivity between    amygdala and medial frontal cortex. The International Journal of    Neuropsychopharmacology 16, 255-260.-   94. Salvador R, Sarro S, Gomar J J, Ortiz-Gill J, Vila F, Capdevila    Builmore E, McKenna P J, Pomarol-Ciotet E (2010). Overall brain    connectivity maps show cortico-subcortical abnormalities in    schizophrenia. Hum. Brain Mapp. 31, 2003-2014.-   95. Lapidus K A, Levitch C F, Perez A M, Brallier J W, Parides M K,    Soieimani L, et al (2014): A Randomized Controlled Trial of    Intranasal Ketamine in Major Depressive Disorder. Biol Psychiatry.-   96. Ebert A, Kolb M, Heller J, Edel M-A, Roser P, BrCine M (2013):    Modulation of interpersonal trust in borderline personality disorder    by intranasal oxytocin and childhood trauma. Soc Neurosci,    8:305-313.-   97. Klackl J, Pfundmair M, Agroskin D, Jonas E (2013): Who is to    blame? Oxytocin promotes nonpersonalistic attributions in response    to a trust betrayal. Biol Psychol. 92:387-394.-   98. Akerlund M, Bossmar T, Brouarcl R, Kostrzewska A, Laudanski T,    Lemancewicz A, at al (1999): Receptor binding of oxytocin and    vasopressin antagonists and inhibitory effects on isolated    myometrium from preterm and term pregnant women, BJOG. An    International Journal of Obstetrics & Gynaecology. 106:1047-1053.-   99. Manning M, Misicka A, Olma A, Bankowski K, Stoev S, Chini B, at    al (2012): Oxytocin and vasopressin agonists and antagonists as    research tools and potential therapeutics, J Neuroendocrinol.    24:609-628.-   100. Parker K J, Buckmaster C L, Schatzberg A F, Lyons D M (2005):    Intranasal oxytocin administration attenuates the ACTH stress    response in monkeys. Psychoneuroendocrinology, 30:924-929.-   101. Calcagnoli F, Meyer N, de Boer S F, Althaus M, Koolhaas J M    (2014): Chronic enhancement of brain oxytocin levels causes enduring    anti-aggressive and pro-social explorative behavioral effects in    male rats. Horm Behav. 65:427-433.-   102. McGregor I S, Bowen M T (2012): Breaking the loop: oxytocin as    a potential treatment for drug addiction. Horm Behan. 61:331-339.-   103. Quintana D S, Guastella A J, Westlye L T, Andreassen O A (in    press): The promise and pitfalls of intranasally administering    psychopharmacological agents for the treatment of psychiatric    disorders. Mol Psychiatry.-   104. McEwen B B (2004): Brain-fluid barriers: relevance for    theoretical controversies regarding vasopressin and oxytocin memory    research. Adv Pharmacol. 50:531-592.-   105. Pardridge W M (1998): CNS drug design based on principles of    blood-brain barrier transport. J Neurochern. 70:1781-1792.-   106. Ejellestad-Paulsen A, Söderberg-Anim C, Lundin S (1995):    Metabolism of vasopressin, oxytocin, and their analogues in the    human gastrointestinal tract. Peptides. 16:1141-1147.-   107. Leng G, Ludwig M (2015): Intranasal oxytocin: myths and    delusions. Biol Psychiatry.-   108. Quintana D S, Alvares G A, Hickie I B, Guastella A J (2015): Do    delivery routes of intranasally administered oxytocin account for    observed effects on social cognition and behavior? A two-level    model. Neurosci Biobehav Rev. 49:182-192.-   109. Quintana D S, Woolley J D (2015): Intranasal oxytocin    mechanisms can be better understood but its effects on social    cognition and behavior are not to be sniffed at. Biol Psychiatry.-   110. Guastella A J, Hickie I B (2015): Oxytocin treatment, circuitry    and autism: a critical review of the literature placing oxytocin    into the autism context. Biol Psychiatry.-   111. Yamasue H (2015): Promising evidence and remaining issues    regarding the clinical application of oxytocin in autism spectrum    disorders, Psychiatry an Neurosci.-   112. Quintana D S, Westlye L T, Rustan ØG, Tesli N Poppy C L, Smevik    H, at al, (2015): Low dose oxytocin delivered intranasally with    Breath Powered device affects social-cognitive behavior: a    randomized 4-way crossover trial with nasal cavity dimension    assessment. Translational Psychiatry, 5:1-9.-   113. Ousdal O, Jensen I, Server A, Hariri A, Nakstad P, Andreassen O    (2008): The human amygdala is involved in general behavioral    relevance detection: evidence from an event-related functional    magnetic resonance imaging Go-NoGo task. Neuroscience. 156:450-455.-   114. Labuschagne I, Phan K L, Wood A, Angstadt M, Chua P, Heinrichs    M, at al. (2010): Oxytocin Attenuates Amygdala Reactivity to Fear in    Generalized Social Anxiety Disorder. Neuropsychopharmacology.    35:2403-2413.-   115. Petrovic P, Kalisch R, Singer T, Dolan R J (2008): Oxytocin    Attenuates Affective Evaluations of Conditioned Faces and Amygdala    Activity. J Neurosci, 28:6607-6615.-   116. Riem M M, Bakermans-Kranenburg M J, Pieper S, Tops M, Boksem M    A, Vermeiren R R, at al. (2011): Oxytocin modulates amygdala,    insula, and inferior frontal gyrus responses to infant crying: a    randomized controlled trial. Biot Psychiatry, 70:291-297.-   117. Riem M M, van Dzendoorn M H, Tops M, Boksem M A, Rombouts S A,    Bakermans-Kranenburg M J (2012): No laughing matter: intranasal    oxytocin administration changes functional brain connectivity during    exposure to infant laughter. Neuropsychopharmacology. 337:1257-1266.-   118. Kemp A H, Guastella A J (2011): The role of oxytocin in human    affect a novel hypothesis. Current Directions in Psychological    Science. 20:222-231.-   119. Bradley M M, Miccoli L, Escrig M A, Lang P J (2008): The pupil    as a measure of emotional arousal and autonomic activation,    Psychophysiology. 45:602-607.-   120. Prehn K, Heekeren H R, Van der Meer E (2011): influence of    affective significance on different levels of processing using pupil    dilation in an analogical reasoning task. Int J Psychophysiol.    79:236-243.-   121, Prehn K, Kazzer P, Lischke A, Heinrichs M, Herpertz S C, Domes    G (2013): Effects of intranasal oxytocin on pupil diiation indicate    increased salience of socioaffective stimuli. Psychophysiology,    50.528-537.-   122. Djupesland P G, Mahmoud R A, Messina J C (2013): Accessing the    brain: the nose may know the way. Journal of Cerebral Blood Flow    &amp;amp; Metabolism. 33:793-794.-   123. Fischi B, Saiat D H Paula E, Albert M, Dieterich M, Haselgrove    C, et al, (2002): Whole brain segmentation: automated labeling of    neuroanatomical structures in the human brain, Neuron. 33:341-355.-   124. Jenkinson M, Bannister P, Brady M, Smith S (2002): Improved    optimization for the robust and accurate linear registration and    motion correction of brain images. Neuroimage. 17:825-841.-   125. Smith S M, Brady J M (1997): SUSAN—A new approach to low level    image processing. International journal of computer vision.    23:45-78.-   126. Beckmann C F, Smith S M (2004): Probabilistic independent    component analysis for functional magnetic resonance imaging,    Medical Imaging, IEEE Transactions on. 23:137-152.-   127. Greve D N, Fischi B (2009): Accurate and robust brain image    alignment using boundary-based registration. Neuroimage, 48:63-72.-   128. Woolrich M W, Ripley B D, Brady M, Smith S M (2001): Temporal    autocorrelation in univariate linear modeling of FIERI data.    Neuroirnage. 14:1370-1386.-   129. Smith S M, Jenkinson M, Woolrich M W, Beckmann C F, Behrens T    E, Johansen-Berg H, et al, (2004): Advances in functional and    structural MR image analysis and implementation as FSL. Neuroimage,    23:S208-S219.-   130, Jeffreys H (1998): The theory of probability, Oxford, UK:    Oxford University Press.-   131. Veinante P, Freund-Mercier M J (1997): Distribution of    oxytocin-and vasopressin-binding sites in the rat extended amygdala    : a histoautoradiographic study. J Comp Neural. 383:305-325.-   132. Insel T R, Shapiro L E (1992): Oxytocin receptor distribution    reflects social organization in monogamous and polygamous voles.    Proceedings of the National Academy of Sciences, 89:5981-5985.-   133. Huber D, Veinante P, Stoop R (2005): Vasopressin and oxytocin    excite distinct neuronal populations in the central amygdala.    Science. 308:245-248.-   134. Knobloch H S, Charlet A, Hoffmann L C, Eliava M, Khrulev S,    Cetin A H, et al. (2012): Evoked axonal oxytocin release in the    central amygdala attenuates fear response. Neuron. 73:553-566.-   135. Thorne R G, Pronk G J, Padmanabhan V, Frey I, W H (2004):    Delivery of insulin-like growth factor-I to the rat brain and spinal    cord along olfactory and trigeminal pathways following intranasal    administration. Neuroscience. 127:481-496.-   136. Ikemoto S (2007): Dopamine reward circuitry: Two projection    systems from the ventral midbrain to the nucleus accumbens-olfactory    tubercle complex, Brain Research Reviews. 56:27-78.-   137. Kang N, Baum M J, Cherry J A (2011): Different Profiles of Main    and Accessory Olfactory Bulb Mitral/Tufted Cell Projections Revealed    in Mice Using an Anterograde Tracer and a Whole-Mount, flattened    Cortex Preparation. Chem Senses. 36:251-260.-   138. Sosulski D L, Bloom M L, Cutforth T, Axel R, Datta S R (2012):    Distinct representations of olfactory information in different    cortical centres. Nature. 472:2132-216.-   139. Neumann I D, Maloumby R, Beiderbeck D I, Lukas M, Landgraf R    (2013): Increased brain and plasma oxytocin after nasal and    peripheral administration in rats and mice.    Psychoneuroendocrinology. 38:1985-1993.-   140. Mens W B, Witter A, Van Wimersma Greidanus T B (1983):    Penetration of neurohypophyseal hormones from plasma into    cerebrospinal fluid (CSF): half-times of disappearance of these    neuropeptides from CSF. Brain Res. 262:143-149.-   141. Medi M E, Connor- Stroud F, Landgraf R, Young U, Parr L A    (2014):

Aerosolized oxytocin increases cerebrospinal fluid oxytocin in rhesusmacaques. Psychoneuroendocrinology, 45:49-57.

-   142, Gouin J-P, Carter C S, Pournajafi-Nazarloo H, Glaser R,    Malarkey W B, Loving T J, et al, (2010): Marital behavior, oxytocin,    vasopressin, and wound healing, Psychoneuroendocrinology.    35:1082-1090.-   143, Rodrigues SM, Saslow LR, Garda N, John OP, Keitner D (2009):    Oxytocin receptor genetic variation relates to empathy and stress    reactivity in humans. Proceedings of the National Academy of    Sciences. 106:21437-21441.

1-71. (canceled)
 72. A delivery device for modulating a condition in ahuman subject comprising: a mouthpiece configured to fit within an oralcavity of the subject and permit the subject to exhale therethrough,whereby exhalation by the subject causes closure of the oropharyngealvelum of the subject; a nosepiece fluidly connected to the mouthpiece,the nosepiece configured to fit within a nostril of the subject, whereinthe device is configured to deliver an exhalation breath of the subjectfrom the mouthpiece, through the nosepiece, and into a first nasalcavity of the subject; and a delivery unit containing oxytocin, theoxytocin comprising oxytocin, non-peptide agonists, and/or non-peptideantagonist thereof, the delivery unit configured to deliver the oxytocinthrough the nosepiece; wherein the oxytocin is delivered through thenosepiece to an upper posterior region, posterior of the nasal valveinnervated by the trigeminal nerve.
 73. The delivery device of claim 72,wherein when the exhalation breath of the subject is delivered throughthe nosepiece into the first nasal cavity of the subject, it acts toestablish a flow through the nasal cavity of the subject around aposterior margin of the subject, and out of a second nasal cavity of thesubject.
 74. The delivery device of claim 73, wherein the exhalationbreath is delivered substantially simultaneously with administration ofthe oxytocin.
 75. The delivery device of claim 72, wherein the oxytocinis a liquid.
 76. The delivery device of claim 75, wherein the oxytocinis delivered as a spray.
 77. The delivery device of claim 72, whereinthe oxytocin is delivered as a powder.
 78. The delivery device of claim77, wherein the oxytocin is delivered as a spray.
 79. The deliverydevice of claim 72, wherein the oxytocin comprises desglycinamideoxytocin.
 80. The delivery device of claim 72, wherein the oxytocincomprises carbetocin.
 81. The delivery device of claim 72, wherein theoxytocin comprises demoxytocin
 82. The delivery device of claim 72,wherein the oxytocin comprises both peptide and non-peptide oxytocin.83. The delivery device of claim 72, wherein less than 24 IU of theoxytocin is delivered.
 84. The delivery device of claim 72, wherein lessthan 15 IU of the oxytocin is delivered.
 85. The delivery device ofclaim 72, wherein less than 12 IU of the oxytocin is delivered.
 86. Thedelivery device of claim 72, wherein less than 10 IU of the oxytocin isdelivered.
 87. The delivery device of claim 72, wherein more than 1 IUof the oxytocin is delivered.
 88. The delivery device of claim 72,wherein more than 2 IU of the oxytocin is delivered.
 89. The deliverydevice of claim 72, wherein more than 4 IU of the oxytocin is delivered.90. A delivery device for modulating a condition in a human subjectcomprising: a mouthpiece configured to fit within an oral cavity of thesubject and permit the subject to exhale therethrough, wherebyexhalation by the subject causes closure of the oropharyngeal velum ofthe subject; a nosepiece fluidly connected to the mouthpiece, thenosepiece having a longitudinal axis and configured to fit within anostril of the subject, the nosepiece comprising a body having a baseportion, a tip, and a projection extending from the base portion,wherein the tip is configured to extend into the nasal valve of thesubject and expand the nasal valve, wherein the device is configured todeliver an exhalation breath of a subject from the mouthpiece, throughthe nosepiece, and into a first nasal cavity of the subject; and adelivery unit containing oxytocin, the oxytocin comprising oxytocin,non-peptide agonists, and/or non-peptide antagonist thereof, thedelivery unit configured to deliver the oxytocin through the nosepiece;wherein the oxytocin is delivered through the nosepiece to an upperposterior region, posterior of the nasal valve innervated by thetrigeminal nerve.
 91. The delivery device of claim 90, wherein when theexhalation breath of the subject is delivered through the nosepiece intothe first nasal cavity of the subject, it acts to establish a flowthrough the nasal cavity of the subject around a posterior margin of thesubject, and out of a second nasal cavity of the subject.
 92. Thedelivery device of claim 91, wherein the exhalation breath is deliveredsubstantially simultaneously with administration of the oxytocin. 93.The delivery device of claim 90, wherein the oxytocin is a liquid. 94.The delivery device of claim 93, wherein the oxytocin is delivered as aspray.
 95. The delivery device of claim 90, wherein the oxytocin is apowder.
 96. The delivery device of claim 96, wherein the oxytocin isdelivered as a spray
 97. The delivery device of claim 90, wherein theoxytocin comprises desglycinamide oxytocin.
 98. The delivery device ofclaim 90, wherein the oxytocin comprises carbetocin.
 99. The deliverydevice of claim 90, wherein the oxytocin comprises demoxytocin
 100. Thedelivery device of claim 90, wherein the oxytocin comprises both peptideand non-peptide oxytocin.
 101. The delivery device of claim 90, whereinless than 24 IU of the oxytocin is delivered.
 102. The delivery deviceof claim 90, wherein less than 15 IU of the oxytocin is delivered. 103.The delivery device of claim 90, wherein less than 12 IU of the oxytocinis delivered.
 104. The delivery device of claim 90, wherein less than 10IU of the oxytocin is delivered.
 105. The delivery device of claim 90,wherein more than 1 IU of the oxytocin is delivered.
 106. The deliverydevice of claim 90, wherein more than 2 IU of the oxytocin is delivered.107. The delivery device of claim 90, wherein more than 4 IU of theoxytocin is delivered.
 108. The delivery device of claim 90, wherein thebody of the nosepiece defines a flow path through the nosepiece. 109.The delivery device of claim 90, wherein the projection forms a portionof the tip of the nosepiece.
 110. The delivery device of claim 90,wherein the projection extends substantially parallel and offset withrespect to the longitudinal axis of the nosepiece.
 111. The deliverydevice of claim 90, wherein the projection comprises a flat element witha first length in the sagittal direction and a second length in thelateral direction, the first length being greater than the secondlength.
 112. The delivery device of claim 111, wherein the first lengthis 1.5 times greater than the second length.
 113. The delivery device ofclaim 111, wherein the first length is 1.7 times greater than the secondlength.
 114. The delivery device of claim 111, wherein the first lengthis 1.9 times greater than the second length.
 115. The delivery device ofclaim 111, wherein the first length is 2 times greater than the secondlength.
 116. The delivery device of claim 111, wherein the projectionincludes a tapering cross-section along the longitudinal extent of theprojection.
 117. The delivery device of claim 111, wherein the firstlength is less than 3 mm.
 118. The delivery device of claim 111, whereinthe first length is less than 1.5 mm.
 119. The delivery device of claim111, wherein the second length is less than 1.5 mm.
 120. The deliverydevice of claim 111, wherein the second length is greater than 0.5 mm.121. The delivery device of claim 90, wherein the projection comprises aflat element comprising a body section with a first length in thesagittal direction and a second length in the lateral direction, and atip section having a third length in the sagittal direction, the firstlength being greater than the third length.
 122. The delivery device ofclaim 90, wherein the base portion of the nosepiece defines asubstantially annular surface which is inclined in relation to thelongitudinal axis of the nosepiece.
 123. The delivery device of claim90, wherein the nosepiece further comprises an outer body part disposedabout the projection.
 124. The device of claim 90, wherein the bodycomprises a plastics material.
 125. The device of claim 123, wherein theouter body part comprises a resilient material, optionally a rubber orelastomeric material.
 126. The device of claim 123, wherein the outerbody part comprises thermoplastic elastomer.
 127. A method formodulating a condition in a human subject comprising: fitting amouthpiece to an oral cavity of the subject; fitting a nosepiece fluidlyconnected to the mouthpiece to a nostril of the subject, the nosepiecehaving a longitudinal axis and comprising a body having a base portion,a tip, and a projection extending from the base portion, the tipconfigured to extend into the nasal valve of the subject and expand thenasal valve; causing an exhalation breath of the subject to flow fromthe mouthpiece, through the nosepiece, and into a first nasal cavity ofthe subject; wherein exhalation by the subject causes closure of theoropharyngeal velum of the subject; and delivering oxytocin from adelivery unit through the nosepiece to an upper region posterior of thenasal valve innervated by the trigeminal nerve, the oxytocin comprisingoxytocin, non-peptide agonists and/or antagonist thereof.
 128. Themethod of claim 127, wherein the exhalation breath of the subject isdelivered through the nosepiece into the first nasal cavity of thesubject, and acts to establish a flow through the nasal cavity of thesubject around a posterior margin of the subject, and out of a secondnasal cavity of the subject.
 129. The method of claim 127, wherein theexhalation breath is delivered substantially simultaneously withadministration of the oxytocin.
 130. The method of claim 127, whereinthe oxytocin is a liquid.
 131. The method of claim 130, wherein theoxytocin is delivered as a spray.
 132. The method of claim 127, whereinthe oxytocin is a powder.
 133. The method of claim 132, wherein theoxytocin is delivered as a spray
 134. The method of claim 127, whereinthe oxytocin comprises desglycinamide oxytocin.
 135. The method of claim127, wherein the oxytocin comprises carbetocin.
 136. The method of claim127, wherein the oxytocin comprises demoxytocin
 137. The method of claim127, wherein the oxytocin comprises both peptide and non-peptideoxytocin.
 138. The method of claim 127, wherein less than 24 IU of theoxytocin is delivered.
 139. The method of claim 127, wherein less than15 IU of the oxytocin is delivered.
 140. The method of claim 127,wherein less than 12 IU of the oxytocin is delivered.
 141. The method ofclaim 127, wherein less than 10 IU of the oxytocin is delivered. 142.The method of claim 127, wherein more than 1 IU of the oxytocin isdelivered.
 143. The method of claim 127, wherein more than 2 IU of theoxytocin is delivered.
 144. The method of claim 127, wherein more than 4IU of the oxytocin is delivered.
 145. The method of claim 127, whereinthe body of the nosepiece defines a flow path through the nosepiece.146. The method of claim 127, wherein the projection forms a portion ofthe tip of the nosepiece.
 147. The method of claim 127, wherein theprojection extends substantially parallel and offset with respect to thelongitudinal axis of the nosepiece.
 148. The method of claim 127,wherein the projection comprises a flat element with a first length inthe sagittal direction and a second length in the lateral direction, thefirst length being greater than the second length.
 149. The method ofclaim 148, wherein the first length is 1.5 times greater than the secondlength.
 150. The method of claim 148, wherein the first length is 1.7times greater than the second length.
 151. The method of claim 148,wherein the first length is 1.9 times greater than the second length.152. The method of claim 148, wherein the first length is 2 timesgreater than the second length.
 153. The method of claim 148, whereinthe projection includes a tapering cross-section along the longitudinalextent of the projection.
 154. The method of claim 148, wherein thefirst length is less than 3 mm.
 155. The method of claim 148, whereinthe first length is less than 1.5 mm.
 156. The method of claim 148,wherein the second length is less than 1.5 mm.
 157. The method of claim148, wherein the second length is greater than 0.5 mm.
 158. The methodof claim 127, wherein the projection comprises a flat element comprisinga body section with a first length in the sagittal direction and asecond length in the lateral direction, and a tip section having a thirdlength in the sagittal direction, the first length being greater thanthe third length.
 159. The method of claim 127, wherein the base portionof the nosepiece defines a substantially annular surface which isinclined in relation to the longitudinal axis of the nosepiece.
 160. Themethod of claim 127, wherein the nosepiece further comprises an outerbody part disposed about the projection.
 161. The method of claim 127,wherein the body comprises a plastics material.
 162. The method of claim160, wherein the outer body part comprises a resilient material. 163.The method of claim 160, wherein the outer body part comprisesthermoplastic elastomer.
 164. The method of claim 127, furthercomprising expanding the nasal valve within the first nasal cavity. 165.The method of claim 164, wherein insertion of the nosepiece into thenasal cavity expands the nasal valve by at least 20 mini ascompared toan unexpanded cross-sectional area of the nasal valve.
 166. The methodof claim 164, wherein insertion of the nosepiece into the nasal cavityexpands the nasal valve by at least 40 mini ascompared to an unexpandedcross-sectional area of the nasal valve.
 167. The method of claim 164,wherein insertion of the nosepiece into the nasal cavity expands thenasal valve by at least 60 mini ascompared to an unexpandedcross-sectional area of the nasal valve.
 168. The method of claim 164,wherein insertion of the nosepiece into the nasal cavity expands thenasal valve by at least 70 mini ascompared to an unexpandedcross-sectional area of the nasal valve.
 169. The method of claim 164,wherein insertion of the nosepiece into the nasal cavity expands thenasal valve by at least 80 mini ascompared to an unexpandedcross-sectional area of the nasal valve.
 170. The method of claim 164,wherein insertion of the nosepiece into the nasal cavity expands thenasal valve by at least 90 mini ascompared to an unexpandedcross-sectional area of the nasal valve.
 171. The method of claim 164,wherein insertion of the nosepiece into the nasal cavity expands thenasal valve by at least 100 mini ascompared to an unexpandedcross-sectional area of the nasal valve.
 172. The method of claim 164,wherein insertion of the nosepiece into the nasal cavity expands thenasal valve by at least 1.5 times the unexpanded cross-sectional area ofthe nasal valve.
 173. The method of claim 164, wherein insertion of thenosepiece into the nasal cavity expands the nasal valve by at least 1.75times the unexpanded cross-sectional area of the nasal valve.
 174. Themethod of claim 164, wherein insertion of the nosepiece into the nasalcavity expands the nasal valve by at least 2 times the unexpandedcross-sectional area of the nasal valve.
 175. The method of claim 164,wherein insertion of the nosepiece into the nasal cavity expands thenasal valve to have a cross-sectional area of at least 100 mm².
 176. Themethod of claim 164, wherein insertion of the nosepiece into the nasalcavity expands the nasal valve to have a cross-sectional area of atleast 120 mm².
 177. The method of claim 164, wherein insertion of thenosepiece into the nasal cavity expands the nasal valve to have across-sectional area of at least 150 mm².
 178. The method of claim 164,wherein insertion of the nosepiece into the nasal cavity expands thenasal valve to have a cross-sectional area of at least 180 mm².
 179. Themethod of claim 164, wherein insertion of the nosepiece into the nasalcavity expands the nasal valve to have a cross-sectional area of atleast 200 mm².
 180. The method of claim 127, wherein the oxytocin isdelivered in a single delivery
 181. The method of claim 127, furthercomprising delivering the oxytocin in a plurality of deliveries. 182.The method of claim 127, wherein the oxytocin is delivered once daily.183. The method of claim 127, wherein the delivery is targeted toprovide for nose-to-brain (N2B) transport of the oxytocin
 184. Themethod of claim 127, wherein the oxytocin is targeted to provide forreduced systemic delivery.
 185. The method of claim 127, wherein thedelivery results in less than a 20% change in the plasma concentrationof vasopressin in the subject, as compared to the plasma concentrationof vasopressin the subject prior to delivery of the oxytocin.
 186. Themethod of claim 127, wherein the delivery results in less than a 10%change in the plasma concentration of vasopressin in the subject, ascompared to the plasma concentration of vasopressin the subject prior todelivery of the oxytocin.
 187. The method of claim 127, wherein thedelivery results in less than a 5% change in the plasma concentration ofvasopressin in the subject, as compared to the plasma concentration ofvasopressin the subject prior to delivery of the oxytocin.
 188. Themethod of claim 127, wherein the delivery results in substantially nochange in the plasma concentration of vasopressin in the subject, ascompared to the plasma concentration of vasopressin the subject prior todelivery of the oxytocin.